Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. Which parameters correlate with endosomal escape vs. cytotoxicity remain elusive. Therefore, we compared the endosomal distribution of six LNP-mRNA formulations in different human cell types. Surprisingly, we found that total uptake is not a good predictor of delivery. Prolonged uptake impaired endosomal acidification and mRNA accumulated in delivery incompetent. We showed that early endocytic/recycling compartments have the highest probability for mRNA escape. Our results change the view of the mechanisms of endosomal escape and could guide the development of LNPs towards higher efficacy and lower cytotoxicity.

Paramasivam, P., Franke, C., Stöter, M., Höijer, A., Bartesaghi, S., Sabirsh, A., Lindfors, L., Yanez Arteta, M., Dahlén, A., Bak, A., Andersson, S., Kalaidzidis, Y., Bickle, M., Zerial, M. Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.  J Cell Biol (2021): e202103003, doi: 10.1083/jcb.202110137

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

Epithelial cells share their apical surfaces to form a tubular lumen in many organs. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen ensuring its anisotropic expansion. These previously unrecognized structures form a pattern reminiscent of the bulkheads of boats, also present in the developing and adult liver. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Belicova, L., Repnik, U., Delpierre, L., Gralinska, E., Seifert, S., Valenzuela, J.I., Morales-Navarrete, H., Franke, C., Räägel, H., Shcherbinina, E., Prikazchikova, T., Koteliansky, V., Martin Vingron, Kalaidzidis, Y., Zatsepin, T., Zerial, M. (2021). Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads. J Cell Biol, 220(10) Art. No. e202103003 (2021) 

An endosomal tether undergoes an entropic collapse to bring vesicles together

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 recruited to PI3P membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.

Murray, D.H., Jahnel, M., Lauer, J., Avellaneda, M.J., Brouilly, N., Cezanne, A., Morales-Navarrete. H, Perini, E.D., Ferguson, C., Lupas, A.N., Kalaidzidis, Y., Parton, R.G., Grill, S.W., Zerial, M. (2016). An endosomal tether undergoes an entropic collapse to bring vesicles together. Nature 537(7618):107-111. 

A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture

We designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from high-resolution microscopy images. It includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We analysed liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. We uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization.

Morales-Navarrete, H., Segovia-Miranda, F., Klukowski, P., Meyer, K., Nonaka, H., Marsico, G., Chernykh, M., Kalaidzidis, A., Zerial M., Kalaidzidis Y. A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture. Elife, 4 Art. No. e11214 (2015)

* joined first author # joined corresponding author

Prasath Paramasivam, Martin Stöter, Eloina Corradi, Irene Dalla Costa, Andreas Höijer, Stefano Bartesaghi, Alan Sabirsh, Lennart Lindfors, Marianna Yanez Arteta, Peter Nordberg, Shalini Andersson, Marie-Laure Baudet, Marc Bickle, Marino Zerial
Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.
RNA, 28(3) 433-446 (2022)
Open Access DOI
Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Prasath Paramasivam✳︎, Christian Franke✳︎, Martin Stöter, Andreas Höijer, Stefano Bartesaghi, Alan Sabirsh, Lennart Lindfors, Marianna Yanez Arteta, Anders Dahlén, Annette Bak, Shalini Andersson, Yannis Kalaidzidis, Marc Bickle, Marino Zerial
Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.
J Cell Biol, 221(2) Art. No. e202110137 (2022)
Open Access PDF DOI
Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.

German Belenguer✳︎, Gianmarco Mastrogiovanni✳︎, Clare Pacini✳︎, Zoe Hall, Anna Dowbaj, Robert Arnes-Benito, Aleksandra Sljukic, Nicole Prior, Sofia Kakava, Charles R. Bradshaw, Susan E Davies, Michele Vacca, Kourosh Saeb-Parsy, Bon-Kyoung Koo, Meritxell Huch
RNF43/ZNRF3 loss predisposes to hepatocellular-carcinoma by impairing liver regeneration and altering the liver lipid metabolic ground-state.
Nat Commun, 13(1) Art. No. 334 (2022)
Open Access DOI
RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer.

Lenka Belicova, Urska Repnik, Julien Delpierre, Elzbieta Gralinska, Sarah Seifert, José Ignacio Valenzuela, Hernán Morales-Navarrete, Christian Franke, Helin Räägel, Evgeniya Shcherbinina, Tatiana Prikazchikova, Victor Koteliansky, Martin Vingron, Yannis Kalaidzidis, Timofei Zatsepin, Marino Zerial
Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.
J Cell Biol, 220(10) Art. No. e202103003 (2021)
Open Access DOI
Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Nikoleta Kostopoulou✳︎, Sofia Bellou✳︎, Eleni Bagli, Maria Markou, Eleftherios Kostaras, Marko Hyvönen, Yiannis Kalaidzidis, Angelos Papadopoulos, Varvara Chalmantzi, Athena Kyrkou, Ekaterini Panopoulou, Theodore Fotsis, Carol Murphy
Embryonic stem cells are devoid of macropinocytosis, a trafficking pathway for activin A in differentiated cells.
J Cell Sci, 134(13) Art. No. jcs246892 (2021)
Ligand-receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A-receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.

Anastasia Solomatina, Yannis Kalaidzidis, Alice Cezanne, Karen Soans, Caren Norden, Marino Zerial, Ivo F. Sbalzarini
Particle-based Segmentation of Extended Objects on Curved Biological Membranes.
In: ISBI 2021, IEEE International Symposium on Biomedical Imaging (ISBI) (2021), Piscataway, N.J., IEEE (2021), 1150-1154
We present a novel method for model-based segmentation of extended, blob-like objects on curved surfaces. Our method addresses several challenges arising when imaging curved biological membrane, such as out-of-membrane signal and geometry-induced background variations. We use a particle-based reconstruction of the membrane geometry, moment-conserving intensity interpolation from pixels to surface particles, and model-based in-surface segmentation. Our method denoises and deconvolves images, corrects for background variations, and quantifies the number, size, and intensity of segmented objects. We benchmark the accuracy of the method and present two applications to (1) neuroepithelial focal adhesion sites during optic cup morphogenesis in zebrafish and (2) reconstituted membrane domains bearing the small GTPase Rab5 on spherical beads.

Janelle Lauer, Marino Zerial
Profiling Structural Alterations During Rab5 Nucleotide Exchange by HDX-MS.
Methods Mol Biol, 2293 69-89 (2021)
Hydrogen deuterium exchange mass spectrometry (HDX-MS) gives insight into the structure of proteins. By monitoring the rate of exchange of the amide hydrogens on the protein backbone with deuterium atoms in the solvent, one can determine if a given region is highly structured or dynamic, map binding sites of interacting molecules or determine if a binding event is associated with allosteric structural alterations in a protein. Herein, we illustrate the use of this technique to monitor the nucleotide exchange process in Rab5, using the guanine nucleotide exchange factor (GEF)-effector complex, Rabex5:Rabaptin5. By simultaneously monitoring the HDX in Rab5, Rabex5 and Rabaptin5, we can directly visualize nucleotide exchange in Rab5, gain mechanistic insights into the exchange reaction and, by witnessing the transfer of Rab5 from Rabex5 to Rabaptin5, provide direct evidence for the positive feedback loop generated by a GEF-effector complex. HDX-MS can be used to monitor a variety of Rab protein-effector and -regulator interactions and be widely applied to other enzymatic processes as well.

André Scholich, Simon Syga, Hernán Morales-Navarrete, Fabián Segovia-Miranda, Hidenori Nonaka, Kirstin Meyer, Walter de Back, Lutz Brusch, Yannis Kalaidzidis, Marino Zerial, Frank Jülicher, Benjamin Friedrich
Quantification of nematic cell polarity in three-dimensional tissues.
PLoS Comput Biol, 16(12) Art. No. 1008412 (2020)
Open Access DOI
How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarette et al., eLife 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues and in-vitro organoids.

Alice Cezanne, Janelle Lauer, Anastasia Solomatina, Ivo F. Sbalzarini, Marino Zerial
A non-linear system patterns Rab5 GTPase on the membrane.
Elife, 9 Art. No. e54434 (2020)
Open Access PDF DOI
Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a 'handover' of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

Jens Karschau, André Scholich, Jonathan Wise, Hernán Morales-Navarrete, Yannis Kalaidzidis, Marino Zerial, Benjamin Friedrich
Resilience of three-dimensional sinusoidal networks in liver tissue.
PLoS Comput Biol, 16(6) Art. No. e1007965 (2020)
Open Access DOI
Can three-dimensional, microvasculature networks still ensure blood supply if individual links fail? We address this question in the sinusoidal network, a plexus-like microvasculature network, which transports nutrient-rich blood to every hepatocyte in liver tissue, by building on recent advances in high-resolution imaging and digital reconstruction of adult mice liver tissue. We find that the topology of the three-dimensional sinusoidal network reflects its two design requirements of a space-filling network that connects all hepatocytes, while using shortest transport routes: sinusoidal networks are sub-graphs of the Delaunay graph of their set of branching points, and also contain the corresponding minimum spanning tree, both to good approximation. To overcome the spatial limitations of experimental samples and generate arbitrarily-sized networks, we developed a network generation algorithm that reproduces the statistical features of 0.3-mm-sized samples of sinusoidal networks, using multi-objective optimization for node degree and edge length distribution. Nematic order in these simulated networks implies anisotropic transport properties, characterized by an empirical linear relation between a nematic order parameter and the anisotropy of the permeability tensor. Under the assumption that all sinusoid tubes have a constant and equal flow resistance, we predict that the distribution of currents in the network is very inhomogeneous, with a small number of edges carrying a substantial part of the flow-a feature known for hierarchical networks, but unexpected for plexus-like networks. We quantify network resilience in terms of a permeability-at-risk, i.e., permeability as function of the fraction of removed edges. We find that sinusoidal networks are resilient to random removal of edges, but vulnerable to the removal of high-current edges. Our findings suggest the existence of a mechanism counteracting flow inhomogeneity to balance metabolic load on the liver.

Punit Saraon, Jamie Snider, Yannis Kalaidzidis, Leanne E Wybenga-Groot, Konstantin Weiss, Ankit Rai, Nikolina Radulovich, Luka Drecun, Nika Vučković, Adriana Vučetić, Victoria Wong, Brigitte Thériault, Nhu-An Pham, Jin H Park, Alessandro Datti, Jenny Wang, Shivanthy Pathmanathan, Farzaneh Aboualizadeh, Anna Lyakisheva, Zhong Yao, Yuhui Wang, Babu Joseph, Ahmed Aman, Michael F Moran, Michael Prakesch, Gennady Poda, Richard Marcellus, David Uehling, Miroslav Samaržija, Marko Jakopović, Ming-Sound Tsao, Frances A Shepherd, Adrian Sacher, Natasha Leighl, Anna Akhmanova, Rima Al-Awar, Marino Zerial, Igor Stagljar
A drug discovery platform to identify compounds that inhibit EGFR triple mutants.
Nat Chem Biol, 16(5) 577-586 (2020)
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

Milena Schuhmacher, Andreas T Grasskamp, Pavel Barahtjan, Nicolai Wagner, Benoit Lombardot, Jan Simon Schuhmacher, Pia Sala, Annett Lohmann, Ian Henry, Andrej Shevchenko, Ünal Coskun, Alexander M Walter#, André Nadler#
Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities.
Proc Natl Acad Sci U.S.A., 117(14) 7729-7738 (2020)
Open Access DOI
Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

Takashi Namba#, Judit Dóczi, Anneline Pinson, Lei Xing, Nereo Kalebic, Michaela Wilsch-Bräuninger, Katherine S. Long, Samir Vaid, Janelle Lauer, Aliona Bogdanova, Barbara Borgonovo, Anna Shevchenko, Patrick Keller, David N. Drechsel, Teymuras V. Kurzchalia, Pauline Wimberger, Christos Chinopoulos, Wieland Huttner#
Human-Specific ARHGAP11B Acts in Mitochondria to Expand Neocortical Progenitors by Glutaminolysis.
Neuron, 105(5) 867-881 (2020)
The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

Kirstin Meyer, Hernán Morales-Navarrete, Sarah Seifert, Michaela Wilsch-Braeuninger, Uta Dahmen, Elly M. Tanaka, Lutz Brusch, Yannis Kalaidzidis, Marino Zerial
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration.
Mol Syst Biol, 16(2) Art. No. e8985 (2020)
Open Access DOI
The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase in F-actin and phospho-myosin, to compensate an overload of bile acids. These changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to apical F-actin-rich regions and translocates to the nucleus in dependence of the integrity of the actin cytoskeleton. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signaling, we explained this behavior by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status.

Priyanka Sharma, Sameena Parveen, Lekha V Shah, Madhumita Mukherjee, Yannis Kalaidzidis, Anthony J Kozielski, Roberto Rosato, Jenny C Chang, Sunando Datta
SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.
J Cell Biol, 219(1) Art. No. e201812098 (2020)
Open Access DOI
A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

Fabián Segovia-Miranda, Hernán Morales-Navarrete, Michael Kücken, Vincent Moser, Sarah Seifert, Urska Repnik, Fabian Rost, Mario Brosch, Alexander Hendricks, Sebastian Hinz, Christoph Röcken, Dieter Lütjohann, Yannis Kalaidzidis, Clemens Schafmayer, Lutz Brusch, Jochen Hampe#, Marino Zerial#
Three-dimensional spatially resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression.
Nat Med, 25(12) 1885-1893 (2019)
Early disease diagnosis is key to the effective treatment of diseases. Histopathological analysis of human biopsies is the gold standard to diagnose tissue alterations. However, this approach has low resolution and overlooks 3D (three-dimensional) structural changes resulting from functional alterations. Here, we applied multiphoton imaging, 3D digital reconstructions and computational simulations to generate spatially resolved geometrical and functional models of human liver tissue at different stages of non-alcoholic fatty liver disease (NAFLD). We identified a set of morphometric cellular and tissue parameters correlated with disease progression, and discover profound topological defects in the 3D bile canalicular (BC) network. Personalized biliary fluid dynamic simulations predicted an increased pericentral biliary pressure and micro-cholestasis, consistent with elevated cholestatic biomarkers in patients' sera. Our spatially resolved models of human liver tissue can contribute to high-definition medicine by identifying quantitative multiparametric cellular and tissue signatures to define disease progression and provide new insights into NAFLD pathophysiology.

Janelle Lauer, Sandra Segeletz, Alice Cezanne, Giambattista Guaitoli, Francesco Raimondi, Marc Gentzel, Vikram Alva, Michael Habeck, Yannis Kalaidzidis, Marius Ueffing, Andrei N Lupas, Christian Johannes Gloeckner, Marino Zerial
Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding.
Elife, 8 Art. No. e46302 (2019)
Open Access DOI
Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

Kseniia Nikitina, Sandra Segeletz, Michael Kuhn, Yannis Kalaidzidis, Marino Zerial
Basic Phenotypes of Endocytic System Recognized by Independent Phenotypes Analysis of a High-throughput Genomic Screen.
In: Proceedings of the 2019 3rd International Conference on Computational Biology and Bioinformatics (2019), New York, ACM (2019), 69-75
High-content screens (HCS) using chemical and genomic interference based on light microscopy and quantitative image analysis yielded a large amount of multi-parametric (MP) phenotypic data. Such data-sets hold great promise for the understanding of cellular mechanisms by systems biology. However, extracting functional information from data-sets, such as links between cellular processes and the functions of unknown genes, remains challenging. The limitation of HCS analysis lies in the complexity of cellular organization. Here, we assumed that cellular processes have a modular structure, and deconvolved the MP data into separate signals from different cellular modules by Blind Source Separation. We applied a combination of quantitative MP image analysis (QMPIA) and Independent Component Analysis (ICA) to an image-based HCS of endocytosis, the process whereby cells uptake molecules from the outside and distribute them to different sub-cellular organelles. We named our approach Independent Phenotypes Analysis (IPA). Phenotypic traits revealed by IPA are interpretable in terms of perturbation of specific endosomal populations (e.g. specific cargo, specific molecular markers) and of specific functional modules (early stages of endocytosis, recycling, cell cycle, etc.). The profile of perturbation of each gene in such basic phenotypic coordinates intrinsically suggest its possible mode of action.

Hernán Morales-Navarrete, Fabián Segovia-Miranda, Marino Zerial, Yannis Kalaidzidis
Prediction of Multiple 3D Tissue Structures Based on Single-Marker Images Using Convolutional Neural Networks.
In: 2019 IEEE International Conference on Image Processing : proceedings : September 22-25, 2019, Taipei International Convention Center (TICC), Taipei, Taiwan (2019), Piscataway, N.J., IEEE (2019), 1361-1365
A quantitative understanding of complex biological systems such as tissues requires reconstructing the structure of the different components of the system. Fluorescence microscopy provides the means to visualize simultaneously several tissue components. However, it can be time consuming and is limited by the number of fluorescent markers that can be used. In this study, we describe a toolbox of algorithms based on convolutional neural networks for the prediction of 3D tissue structures by learning features embedded within single-marker images. As proof of principle, we aimed to predict the network of bile canaliculi (BC) in liver tissue using images of the cortical actin mesh as input. The actin meshwork has a characteristic organization in specific cellular domains, such as BC. However, the use of manually selected features from images of actin is not sufficient to properly reconstruct BC structure. Our deep learning framework showed a remarkable accuracy for the prediction of BC network and was successfully adapted (i.e. transfer learning) to predict the sinusoidal network. This approach allows for a complete reconstruction of tissue microarchitecture using a single fluorescent marker.

Christian Franke, Urska Repnik, Sandra Segeletz, Nicolas Brouilly, Yannis Kalaidzidis, Jean-Marc Verbavatz, Marino Zerial
Correlative single-molecule localization microscopy and electron tomography reveals endosome nanoscale domains.
Traffic, 20(8) 601-617 (2019)
Open Access DOI
Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Hernán Morales-Navarrete✳︎, Hidenori Nonaka✳︎, André Scholich✳︎, Fabián Segovia-Miranda✳︎, Walter de Back, Kirstin Meyer, Roman L Bogorad, Victor Koteliansky, Lutz Brusch, Yannis Kalaidzidis#, Frank Jülicher#, Benjamin Friedrich#, Marino Zerial#
Liquid-crystal organization of liver tissue.
Elife, 8 Art. No. e44860 (2019)
Open Access DOI
Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images of mouse liver tissue and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells, since silencing Integrin-β1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

Beatrice Senigagliesi, Carlotta Penzo, Luisa Ulloa Severino, Riccardo Maraspini, Sara Petrosino, Hernán Morales-Navarrete, Enrico Pobega, Elena Ambrosetti, Pietro Parisse, Silvia Pegoraro, Guidalberto Manfioletti, Loredana Casalis, Riccardo Sgarra
The High Mobility Group A1 (HMGA1) Chromatin Architectural Factor Modulates Nuclear Stiffness in Breast Cancer Cells.
Int J Mol Sci, 20(11) Art. No. 2733 (2019)
Open Access DOI
Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

Livia Goto-Silva, Marisa P. McShane, Sara Salinas, Yannis Kalaidzidis, Giampietro Schiavo, Marino Zerial
Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome.
Sci Rep, 9(1) Art. No. 2433 (2019)
Open Access DOI
Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

Egon Deyaert, Margaux Leemans, Ranjan Kumar Singh, Rodrigo Gallardo, Jan Steyaert, Arjan Kortholt, Janelle Lauer#, Wim Versées#
Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein.
Biochem J, 476(1) 51-66 (2019)
The LRR (leucine-rich repeat)-Roc (Ras of complex proteins)-COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer-monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5'-diphosphate)-bound states and monomeric in the GTP (guanosine-5'-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR-Roc-COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the 'Roc dimerization loop'. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, Sian Culley, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, Ricardo Henriques, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W Myers
Content-aware image restoration: pushing the limits of fluorescence microscopy.
Nat Methods, 15(12) 1090-1097 (2018)
Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

Hiroo Tanaka, Mitsunobu Imasato, Yuji Yamazaki, Kengo Matsumoto, Koshi Kunimoto, Julien Delpierre, Kirstin Meyer, Marino Zerial, Naho Kitamura, Mitsuhiro Watanabe, Atsushi Tamura, Sachiko Tsukita
Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.
J Hepatol, 69(6) 1308-1316 (2018)
Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative.

Oskar Knittelfelder, Sofia Traikov, Olga Vvedenskaya, Andrea Schuhmann, Sandra Segeletz, Anna Shevchenko, Andrej Shevchenko
Shotgun Lipidomics Combined with Laser Capture Microdissection: A Tool To Analyze Histological Zones in Cryosections of Tissues.
Anal Chem, 90(16) 9868-9878 (2018)
Shotgun analysis provides a quantitative snapshot of the lipidome composition of cells, tissues, or model organisms; however, it does not elucidate the spatial distribution of lipids. Here we demonstrate that shotgun analysis could quantify low-picomole amounts of lipids isolated by laser capture microdissection (LCM) of hundred micrometer-sized histological zones visualized at the cryosections of tissues. We identified metabolically distinct periportal (pp) and pericentral (pc) zones by immunostaining of 20 μm thick cryosections of a healthy mouse liver. LCM was used to ablate, catapult, and collect the tissue material from 10 to 20 individual zones covering a total area of 0.3-0.5 mm2 and containing ca. 500 cells. Top-down shotgun profiling relying upon computational stitching of 61 targeted selective ion monitoring ( t-SIM) spectra quantified more than 200 lipid species from 17 lipid classes including glycero- and glycerophospholipids, sphingolipids, cholesterol esters, and cholesterol. Shotgun LCM revealed the overall commonality of the full lipidome composition of pp and pc zones along with significant ( p < 0.001) difference in the relative abundance of 13 lipid species. Follow-up proteomics analyses of pellets recovered from an aqueous phase saved after the lipid extraction identified 13 known and 7 new protein markers exclusively present in pp or in pc zones and independently validated the specificity of their visualization, isolation, and histological assignment.

FoSheng Hsu, Stephanie Spannl, Charles Ferguson, Anthony Hyman, Robert G. Parton, Marino Zerial
Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria.
Elife, 7 Art. No. e32282 (2018)
Open Access DOI
Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS.

Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena
A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.
Sci Rep, 7 Art. No. 23 (2017)
Open Access PDF DOI
Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Simone Pigolotti, Izaak Neri, Édgar Roldán, Frank Jülicher
Generic Properties of Stochastic Entropy Production.
Phys Rev Lett, 119(14) Art. No. 140604 (2017)
We derive an Itô stochastic differential equation for entropy production in nonequilibrium Langevin processes. Introducing a random-time transformation, entropy production obeys a one-dimensional drift-diffusion equation, independent of the underlying physical model. This transformation allows us to identify generic properties of entropy production. It also leads to an exact uncertainty equality relating the Fano factor of entropy production and the Fano factor of the random time, which we also generalize to non-steady-state conditions.

Meik Dörpinghaus, Édgar Roldán, Izaak Neri, Heinrich Meyr, Frank Jülicher
An information theoretic analysis of sequential decision-making
In: 2017 IEEE International Symposium on Information Theory (ISIT) : 25-30 June 2017 (2017)(Eds.) Gerhard Kramer, Piscataway, N.J., IEEE (2017), 3050-3054
We provide a novel analysis of Wald's sequential probability ratio test based on information theoretic measures for symmetric thresholds, symmetric noise, and equally likely hypotheses. This test is optimal in the sense that it yields the minimum mean decision time. To analyze the decision-making process we consider information densities, which represent the stochastic information content of the observations yielding a stochastic termination time of the test. Based on this, we show that the conditional probability to decide for hypothesis H1 (or the counter-hypothesis H0) given that the test terminates at time instant k is independent of time k. An analogous property has been found for a continuous-time first passage problem with two absorbing boundaries in the contexts of non-equilibrium statistical physics and communication theory. Moreover, we study the evolution of the mutual information between the binary variable to be tested and the output of the Wald test. Notably, we show that the decision time of the Wald test contains no information on which hypothesis is true beyond the decision outcome

Yannis Kalaidzidis
Fluorescence Microscopy Noise Model: Estimation of Poisson Noise Parameters from Snap-Shot Image
In: Proceedings of the 2017 International Conference on Bioinformatics & Computational Biology : BIOCOMP 2017 (2017)(Eds.) Hamid R Arabnia, United States, CSREA Press (2017), 63-66

J Gray Camp✳︎, Keisuke Sekine✳︎, Tobias Gerber, Henry Loeffler-Wirth, Hans Binder, Malgorzata Gac, Sabina Kanton, Jorge Kageyama, Georg Damm, Daniel Seehofer, Lenka Belicova, Marc Bickle, Rico Barsacchi, Ryo Okuda, Emi Yoshizawa, Masashi Kimura, Hiroaki Ayabe, Hideki Taniguchi, Takanori Takebe#, Barbara Treutlein#
Multilineage communication regulates human liver bud development from pluripotency.
Nature, 546(7659) 533-538 (2017)
Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Christoph Metzendorf✳︎, Anja Zeigerer✳︎, Sarah Seifert, Richard Sparla, Bahar Najafi, François Canonne-Hergaux, Marino Zerial, Martina Muckenthaler
Acute loss of the hepatic endo-lysosomal system in vivo causes compensatory changes in iron homeostasis.
Sci Rep, 7(1) Art. No. 4023 (2017)
Open Access DOI
Liver cells communicate with the extracellular environment to take up nutrients via endocytosis. Iron uptake is essential for metabolic activities and cell homeostasis. Here, we investigated the role of the endocytic system for maintaining iron homeostasis. We specifically depleted the small GTPase Rab5 in the mouse liver, causing a transient loss of the entire endo-lysosomal system. Strikingly, endosome depletion led to a fast reduction of hepatic iron levels, which was preceded by an increased abundance of the iron exporter ferroportin. Compensatory changes in livers of Rab5-depleted mice include increased expression of transferrin receptor 1 as well as reduced expression of the iron-regulatory hormone hepcidin. Serum iron indices (serum iron, free iron binding capacity and total iron binding capacity) in Rab5-KD mice were increased, consistent with an elevated splenic and hepatic iron export. Our data emphasize the critical importance of the endosomal compartments in hepatocytes to maintain hepatic and systemic iron homeostasis in vivo. The short time period (between day four and five) upon which these changes occur underscore the fast dynamics of the liver iron pool.

Kirstin Meyer, Oleksandr Ostrenko, George Bourantas, Hernán Morales-Navarrete, Natalie Porat-Shliom, Fabián Segovia-Miranda, Hidenori Nonaka, Ali Ghaemi, Jean-Marc Verbavatz, Lutz Brusch, Ivo F. Sbalzarini, Yannis Kalaidzidis, Roberto Weigert, Marino Zerial
A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule.
Cell Syst, 4(3) 277-290 (2017)
Open Access PDF DOI
Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Izaak Neri, Édgar Roldán, Frank Jülicher
Statistics of Infima and Stopping Times of Entropy Production and Applications to Active Molecular Processes
Physical Review X, 7(1) Art. No. 011019 (2017)
Open Access PDF DOI
We study the statistics of infima, stopping times, and passage probabilities of entropy production in nonequilibrium steady states, and we show that they are universal. We consider two examples of stopping times: first-passage times of entropy production and waiting times of stochastic processes, which are the times when a system reaches a given state for the first time. Our main results are as follows: (i) The distribution of the global infimum of entropy production is exponential with mean equal to minus Boltzmann's constant; (ii) we find exact expressions for the passage probabilities of entropy production; (iii) we derive a fluctuation theorem for stopping-time distributions of entropy production. These results have interesting implications for stochastic processes that can be discussed in simple colloidal systems and in active molecular processes. In particular, we show that the timing and statistics of discrete chemical transitions of molecular processes, such as the steps of molecular motors, are governed by the statistics of entropy production. We also show that the extreme-value statistics of active molecular processes are governed by entropy production; for example, we derive a relation between the maximal excursion of a molecular motor against the direction of an external force and the infimum of the corresponding entropy-production fluctuations. Using this relation, we make predictions for the distribution of the maximum backtrack depth of RNA polymerases, which follow from our universal results for entropy-production infima.

Kamil Jastrzębski, Daria Zdżalik-Bielecka, Agnieszka Mamińska, Yannis Kalaidzidis, C Hellberg, Marta Miaczynska
Multiple routes of endocytic internalization of PDGFRβ contribute to PDGF-induced STAT3 signaling.
J Cell Sci, 130(3) 577-589 (2017)
Platelet-derived growth factor receptor β (PDGFRβ) is a receptor tyrosine kinase which upon activation by PDGF-BB stimulates cell proliferation, migration and angiogenesis. Ligand binding induces intracellular signaling cascades but also internalization of the receptor, eventually resulting in its lysosomal degradation. However, endocytic trafficking of receptors often modulates their downstream signaling. We previously reported that internalization of PDGFRβ occurs via dynamin-dependent and -independent pathways but their further molecular determinants remained unknown. Here we show that, in human fibroblasts expressing endogenous PDGFRβ and stimulated with 50 ng/ml PDGF-BB, ligand-receptor uptake proceeds via the parallel routes of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CME involves the canonical AP2 complex as a clathrin adaptor, while CIE requires RhoA-ROCK, Cdc42 and galectin-3, the latter indicating lectin-mediated internalization via clathrin-independent carriers (CLICs). Although different uptake routes appear to be partly interdependent, they cannot fully substitute for each other. Strikingly, inhibition of any internalization mechanism impaired activation of STAT3 but not of other downstream effectors of PDGFRβ. Our data indicate that multiple routes of internalization of PDGFRβ contribute to a transcriptional and mitogenic response of cells to PDGF.

Anja Zeigerer, Anne Wuttke, Giovanni Marsico, Sarah Seifert, Yannis Kalaidzidis, Marino Zerial
Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.
Exp Cell Res, 350(1) 242-252 (2017)
Open Access PDF DOI
Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro.

Izaak Neri, Fernando Lucas Metz
Eigenvalue Outliers of Non-Hermitian Random Matrices with a Local Tree Structure.
Phys Rev Lett, 117(22) Art. No. 224101 (2016)
Spectra of sparse non-Hermitian random matrices determine the dynamics of complex processes on graphs. Eigenvalue outliers in the spectrum are of particular interest, since they determine the stationary state and the stability of dynamical processes. We present a general and exact theory for the eigenvalue outliers of random matrices with a local tree structure. For adjacency and Laplacian matrices of oriented random graphs, we derive analytical expressions for the eigenvalue outliers, the first moments of the distribution of eigenvector elements associated with an outlier, the support of the spectral density, and the spectral gap. We show that these spectral observables obey universal expressions, which hold for a broad class of oriented random matrices.

Veronica Astro, Diletta Tonoli, Sara Chiaretti, Sabrina Badanai, Kristyna Sala, Marino Zerial, Ivan de Curtis
Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover.
Sci Rep, 6 Art. No. 33653 (2016)
Open Access PDF DOI
Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin β1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins, and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes.

David Murray, Marcus Jahnel, Janelle Lauer, Mario Avellaneda, Nicolas Brouilly, Alice Cezanne, Hernán Morales-Navarrete, Enrico Perini, Charles Ferguson, Andrei N Lupas, Yannis Kalaidzidis, Robert G. Parton, Stephan W. Grill, Marino Zerial
An endosomal tether undergoes an entropic collapse to bring vesicles together.
Nature, 537(7618) 107-111 (2016)
An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.

Hassan Mziaut, Bernard Mulligan, Peter Hoboth, Oliver Otto, Anna Ivanova, Maik Herbig, Desiree Schumann, Tobias Hildebrandt, J Dehghany, Anke Sönmez, Carla Münster, M Meyer-Hermann, Jochen Guck, Yannis Kalaidzidis, Michele Solimena
The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512.
Mol Metab, 5(8) 656-668 (2016)
Open Access PDF DOI
Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis.

Hernán Morales-Navarrete, Hidenori Nonaka, Fabián Segovia-Miranda, Marino Zerial, Yannis Kalaidzidis
Automatic recognition and characterization of different non-parenchymal cells in liver tissue.
In: 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI) (2016), Piscataway, N.J., IEEE (2016), 536-540

Hao Yin, Roman L Bogorad, Carmen Barnes, Stephen Walsh, Iris Zhuang, Hidenori Nonaka, Vera M Ruda, Satya Kuchimanchi, Lubomir Nechev, Akin Akinc, Wen Xue, Marino Zerial, Robert Langer, Daniel G Anderson, Victor Koteliansky
RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver.
J Hepatol, 64(4) 899-907 (2016)
The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models.

Gregory A O'Sullivan, Peter Jedlicka, Hong-Xing Chen, Heba Kalbouneh, Angelo Ippolito, Thomas Deller, Ralph A Nawrotzki, Jochen Kuhse, Yannis Kalaidzidis, Joachim Kirsch, Stephan W Schwarzacher, Heinrich Betz
Forebrain-specific loss of synaptic GABAA receptors results in altered neuronal excitability and synaptic plasticity in mice.
Mol Cell Neurosci, 72 101-113 (2016)
Mutations that result in the defective trafficking of γ2 subunit containing GABAA receptors (γ2-GABAARs) are known to reduce synaptic inhibition. Whether perturbed clustering of non-mutated GABAARs similarly reduces synaptic inhibition in vivo is less clear. In this study we provide evidence that the loss of postsynaptic γ2-GABAARs upon postnatal ablation of gephyrin, the major scaffolding protein of inhibitory postsynapses, from mature principal neurons within the forebrain results in reduced induction of long-term potentiation (LTP) and impaired network excitability within the hippocampal dentate gyrus. The preferential reduction in not only synaptic γ2-GABAAR cluster number at dendritic sites but also the decrease in γ2-GABAAR density within individual clusters at dendritic inhibitory synapses suggests that distal synapses are more sensitive to the loss of gephyrin expression than proximal synapses. The fact that these mice display behavioural features of anxiety and epilepsy emphasises the importance of postsynaptic γ2-GABAAR clustering for synaptic inhibition.

Kirstin Meyer
An in vivo investigation on the mechanosensory function of the mouse liver
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)

Roberto Villaseñor, Yannis Kalaidzidis, Marino Zerial
Signal processing by the endosomal system.
Curr Opin Cell Biol, 39 53-60 (2016)
Open Access PDF DOI
Cells need to decode chemical or physical signals from their environment in order to make decisions on their fate. In the case of signalling receptors, ligand binding triggers a cascade of chemical reactions but also the internalization of the activated receptors in the endocytic pathway. Here, we highlight recent studies revealing a new role of the endosomal network in signal processing. The diversity of entry pathways and endosomal compartments is exploited to regulate the kinetics of receptor trafficking, and interactions with specific signalling adaptors and effectors. By governing the spatio-temporal distribution of signalling molecules, the endosomal system functions analogously to a digital-analogue computer that regulates the specificity and robustness of the signalling response.

Michael Kuhn, Ivica Letunic, Lars Juhl Jensen, Peer Bork
The SIDER database of drugs and side effects.
Nucleic Acids Res, 44(D1) 1075-1079 (2016)
Open Access DOI
Unwanted side effects of drugs are a burden on patients and a severe impediment in the development of new drugs. At the same time, adverse drug reactions (ADRs) recorded during clinical trials are an important source of human phenotypic data. It is therefore essential to combine data on drugs, targets and side effects into a more complete picture of the therapeutic mechanism of actions of drugs and the ways in which they cause adverse reactions. To this end, we have created the SIDER ('Side Effect Resource', http://sideeffects.embl.de) database of drugs and ADRs. The current release, SIDER 4, contains data on 1430 drugs, 5880 ADRs and 140 064 drug-ADR pairs, which is an increase of 40% compared to the previous version. For more fine-grained analyses, we extracted the frequency with which side effects occur from the package inserts. This information is available for 39% of drug-ADR pairs, 19% of which can be compared to the frequency under placebo treatment. SIDER furthermore contains a data set of drug indications, extracted from the package inserts using Natural Language Processing. These drug indications are used to reduce the rate of false positives by identifying medical terms that do not correspond to ADRs.

Damian Szklarczyk, Alberto Santos, Christian von Mering, Lars Juhl Jensen, Peer Bork, Michael Kuhn
STITCH 5: augmenting protein-chemical interaction networks with tissue and affinity data.
Nucleic Acids Res, 44(D1) 380-384 (2016)
Open Access DOI
Interactions between proteins and small molecules are an integral part of biological processes in living organisms. Information on these interactions is dispersed over many databases, texts and prediction methods, which makes it difficult to get a comprehensive overview of the available evidence. To address this, we have developed STITCH ('Search Tool for Interacting Chemicals') that integrates these disparate data sources for 430 000 chemicals into a single, easy-to-use resource. In addition to the increased scope of the database, we have implemented a new network view that gives the user the ability to view binding affinities of chemicals in the interaction network. This enables the user to get a quick overview of the potential effects of the chemical on its interaction partners. For each organism, STITCH provides a global network; however, not all proteins have the same pattern of spatial expression. Therefore, only a certain subset of interactions can occur simultaneously. In the new, fifth release of STITCH, we have implemented functionality to filter out the proteins and chemicals not associated with a given tissue. The STITCH database can be downloaded in full, accessed programmatically via an extensive API, or searched via a redesigned web interface at http://stitch.embl.de.

Jaime Huerta-Cepas, Damian Szklarczyk, Kristoffer Forslund, Helen Cook, Davide Heller, Mathias C Walter, Thomas Rattei, Daniel R Mende, Shinichi Sunagawa, Michael Kuhn, Lars Juhl Jensen, Christian von Mering, Peer Bork
eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences.
Nucleic Acids Res, 44(D1) 286-293 (2016)
Open Access DOI
eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. eggNOG v4.5 is available at http://eggnog.embl.de.

Jon M Carthy, Martin Stöter, Claudia Bellomo, Michael Vanlandewijck, Angelos Heldin, Anita Morén, Dimitris Kardassis, Timothy C Gahman, Andrew K Shiau, Marc Bickle, Marino Zerial, Carl-Henrik Heldin, Aristidis Moustakas
Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation.
Sci Rep, 6 Art. No. 29868 (2016)
Open Access PDF DOI
Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer.

Linda Cerofolini, Sabrina Amar, Janelle Lauer, Tommaso Martelli, Marco Fragai, Claudio Luchinat, Gregg B Fields
Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity.
Sci Rep, 6 Art. No. 29511 (2016)
Open Access PDF DOI
Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.

Édgar Roldán, Izaak Neri, Meik Dörpinghaus, Heinrich Meyr, Frank Jülicher
Decision Making in the Arrow of Time.
Phys Rev Lett, 115(25) Art. No. 250602 (2015)
We show that the steady-state entropy production rate of a stochastic process is inversely proportional to the minimal time needed to decide on the direction of the arrow of time. Here we apply Wald's sequential probability ratio test to optimally decide on the direction of time's arrow in stationary Markov processes. Furthermore, the steady-state entropy production rate can be estimated using mean first-passage times of suitable physical variables. We derive a first-passage time fluctuation theorem which implies that the decision time distributions for correct and wrong decisions are equal. Our results are illustrated by numerical simulations of two simple examples of nonequilibrium processes.

Hernán Morales-Navarrete, Fabián Segovia-Miranda, Piotr Klukowski, Kirstin Meyer, Hidenori Nonaka, Giovanni Marsico, Mikhail Chernykh, Alexander Kalaidzidis, Marino Zerial#, Yannis Kalaidzidis#
A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.
Elife, 4 Art. No. e11214 (2015)
Open Access PDF DOI
A prerequisite for the systems biology analysis of tissues is an accurate digital 3D reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness.

Inna Kalaidzidis, Marta Miaczynska, Marta Brewińska-Olchowik, Anna Hupalowska, Charles Ferguson, Robert G. Parton, Yannis Kalaidzidis, Marino Zerial
APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments.
J Cell Biol, 211(1) 123-144 (2015)
Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

Jerome Gilleron, Prasath Paramasivam, Anja Zeigerer, William Querbes, Giovanni Marsico, Cordula Andree, Sarah Seifert, Pablo Amaya, Martin Stöter, Victor Koteliansky, Herbert Waldmann, Kevin Fitzgerald, Yannis Kalaidzidis, Akin Akinc, Martin A Maier, Muthiah Manoharan, Marc Bickle, Marino Zerial
Identification of siRNA delivery enhancers by a chemical library screen.
Nucleic Acids Res, 43(16) 7984-8001 (2015)
Open Access PDF DOI
Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

J Dehghany, Peter Hoboth, Anna Ivanova, Hassan Mziaut, Andreas Müller, Yannis Kalaidzidis, Michele Solimena, M Meyer-Hermann
A Spatial Model of Insulin-Granule Dynamics in Pancreatic β-Cells.
Traffic, 16(8) 797-813 (2015)
Insulin secretion from pancreatic β-cells in response to sudden glucose stimulation is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent-based space-resolved model for SG dynamics in pancreatic β-cells. Using recent experimental data, we consider a single β-cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second-phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that 6 min after glucose stimulation, the 'newcomer' SGs are recruited from a region within less than 600 nm from the PM.

Jovan Tanevski, Ljupčo Todorovski, Yannis Kalaidzidis, Sašo Džeroski
Domain-specific model selection for structural identification of the Rab5-Rab7 dynamics in endocytosis.
BMC Syst Biol, 9 Art. No. 31 (2015)
Open Access DOI
Given its recent rapid development and the central role that modeling plays in the discipline, systems biology clearly needs methods for automated modeling of dynamical systems. Process-based modeling focuses on explanatory models of dynamical systems; it constructs such models from measured time-course data and formalized modeling knowledge. In this paper, we apply process-based modeling to the practically relevant task of modeling the Rab5-Rab7 conversion switch in endocytosis. The task is difficult due to the limited observability of the system variables and the noisy measurements, which pose serious challenges to the process of model selection. To address these issues, we propose a domain-specific model selection criteria that take into account knowledge about the necessary properties of the simulated model behavior.

Anja Zeigerer, Roman L Bogorad, Kirti Sharma, Jerome Gilleron, Sarah Seifert, Susanne Sales, Nikolaus Berndt, Sascha Bulik, Giovanni Marsico, Rochelle C J D'Souza, Naharajan Lakshmanaperumal, Kesavan Meganathan, Karthick Natarajan, Agapios Sachinidis, Andreas Dahl, Hermann-Georg Holzhütter, Andrej Shevchenko, Matthias Mann, Victor Koteliansky, Marino Zerial
Regulation of Liver Metabolism by the Endosomal GTPase Rab5.
Cell Rep, 11(6) 884-892 (2015)
Open Access DOI
The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

Marisa P. McShane, Tim Friedrichson, Angelika Giner, Felix Meyenhofer, Rico Barsacchi, Marc Bickle, Marino Zerial
A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation.
J Biomol Screen, 20(6) 720-728 (2015)
High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.

Peter Hoboth, Andreas Müller, Anna Ivanova, Hassan Mziaut, J Dehghany, Anke Sönmez, Martina Lachnit, M Meyer-Hermann, Yannis Kalaidzidis, Michele Solimena
Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies.
Proc Natl Acad Sci U.S.A., 112(7) 667-676 (2015)
Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.

Roberto Villaseñor, Hidenori Nonaka, Perla Del Conte-Zerial, Yannis Kalaidzidis, Marino Zerial
Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.
Elife, 4 Art. No. e06156 (2015)
Open Access PDF DOI
An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

Damian Szklarczyk, Andrea Franceschini, Stefan Wyder, Kristoffer Forslund, Davide Heller, Jaime Huerta-Cepas, Milan Simonovic, Alexander Roth, Alberto Santos, Kalliopi P Tsafou, Michael Kuhn, Peer Bork, Lars Juhl Jensen, Christian von Mering
STRING v10: protein-protein interaction networks, integrated over the tree of life.
Nucleic Acids Res, 43(D1) 447-452 (2015)
Open Access DOI
The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

Amulya Priya, Inna Kalaidzidis, Yannis Kalaidzidis, David Lambright, Sunando Datta
Molecular insights into rab7-mediated endosomal recruitment of core retromer: deciphering the role of vps26 and vps35.
Traffic, 16(1) 68-84 (2015)
Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans-Golgi network. It consists of two distinct sub-complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub-complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET-based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7-mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub-complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7-mediated recruitment of the core retromer complex.

Yannis Kalaidzidis, Inna Kalaidzidis, Marino Zerial
A Probabilistic Method to Quantify the Colocalization of Markers on Intracellular Vesicular Structures Visualized by Light Microscopy.
In: BAYESIAN INFERENCE AND MAXIMUM ENTROPY METHODS IN SCIENCE AND ENGINEERING (MAXENT 2014) (2015)(Eds.) A. Mohammad-Djafari, F. Barbaresco AIP Conference Proceedings ; 1641, Melville, N.Y., AIP (2015), 580-587

Dirk Drasdo, Johannes Bode, Uta Dahmen, Olaf Dirsch, Steven Dooley, Rolf Gebhardt, Ahmed Ghallab, Patricio Godoy, Dieter Häussinger, Seddik Hammad, Stefan Hoehme, Hermann-Georg Holzhütter, Ursula Klingmüller, Lars Kuepfer, Jens Timmer, Marino Zerial, Jan G Hengstler
The virtual liver: state of the art and future perspectives.
Arch Toxicol, 88(12) 2071-2075 (2014)

Mihaela Anitei, Ramu Chenna, Cornelia Czupalla, Milan Esner, Sara Christ, Steffi Lenhard, Kerstin Korn, Felix Meyenhofer, Marc Bickle, Marino Zerial, Bernard Hoflack
A high-throughput siRNA screen identifies genes that regulate mannose 6-phosphate receptor trafficking.
J Cell Sci, 127(23) 5079-5092 (2014)
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.

Norman Gerstner
Endocytic Modulation of Developmental Signaling during Zebrafish Gastrulation
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2014)

Angela Wandinger-Ness, Marino Zerial
Rab proteins and the compartmentalization of the endosomal system.
Cold Spring Harb Perspect Biol, 6(11) Art. No. a022616 (2014)
Of the approximately 70 human Rab GTPases, nearly three-quarters are involved in endocytic trafficking. Significant plasticity in endosomal membrane transport pathways is closely coupled to receptor signaling and Rab GTPase-regulated scaffolds. Here we review current literature pertaining to endocytic Rab GTPase localizations, functions, and coordination with regulatory proteins and effectors. The roles of Rab GTPases in (1) compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes; (2) coordination of individual transport steps from vesicle budding to fusion; (3) effector interactomes; and (4) integration of GTPase and signaling cascades are discussed.

Sandra L Schmid, Alexander Sorkin, Marino Zerial
Endocytosis: Past, Present, and Future.
Cold Spring Harb Perspect Biol, 6(12) Art. No. a022509 (2014)

Marnix Wieffer, Marisa P. McShane, Marino Zerial
Rab Proteins and the Organization of Organelle Membrane Domains.
In: Ras Superfamily Small G Proteins: Biology and Mechanisms : 2. (Eds.) Alfred Wittinghofer,Cham,Springer International Publishing (2014),17-38 Ch. 2

Angela Simeone, Giovanni Marsico, Claudio Collinet, Thierry Galvez, Yannis Kalaidzidis, Marino Zerial, Andreas Beyer
Revealing molecular mechanisms by integrating high-dimensional functional screens with protein interaction data.
PLoS Comput Biol, 10(9) Art. No. e1003801 (2014)
Open Access DOI
Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT). The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets) and network information (IMPACT-modules). Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1) handles multiple profiles per gene, (2) rescues genes with weak phenotypes and (3) accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens.

Enrico Perini, Ramona Schaefer, Martin Stöter, Yannis Kalaidzidis, Marino Zerial
Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations.
Traffic, 15(12) 1366-1389 (2014)
Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.

Milan Esner, Felix Meyenhofer, Michael Kuhn, Melissa Thomas, Yannis Kalaidzidis, Marc Bickle
Development of a Kinetic Assay for Late Endosome Movement.
J Biomol Screen, 19(7) 1070-1078 (2014)
Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

Varadharajan Sundaramurthy, Rico Barsacchi, Mikhail Chernykh, Martin Stöter, Nadine Tomschke, Marc Bickle, Yannis Kalaidzidis, Marino Zerial
Deducing the mechanism of action of compounds identified in phenotypic screens by integrating their multiparametric profiles with a reference genetic screen.
Nat Protoc, 9(2) 474-490 (2014)
Cell-based high-content screens are increasingly used to discover bioactive small molecules. However, identifying the mechanism of action of the selected compounds is a major bottleneck. Here we describe a protocol consisting of experimental and computational steps to identify the cellular pathways modulated by chemicals, and their mechanism of action. The multiparametric profiles from a 'query' chemical screen are used as constraints to select genes with similar profiles from a 'reference' genetic screen. In our case, the query screen is the intracellular survival of mycobacteria and the reference is a genome-wide RNAi screen of endocytosis. The two disparate screens are bridged by an 'intermediate' chemical screen of endocytosis, so that the similarity in the multiparametric profiles between the chemical and the genetic perturbations can generate a testable hypothesis of the cellular pathways modulated by the chemicals. This approach is not assay specific, but it can be broadly applied to various quantitative, multiparametric data sets. Generation of the query system takes 3-6 weeks, and data analysis and integration with the reference data set takes an 3 additional weeks.

Roman L Bogorad, Hao Yin, Anja Zeigerer, Hidenori Nonaka, Vera M Ruda, Marino Zerial, Daniel G Anderson#, Victor Koteliansky#
Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice.
Nat Commun, 5 Art. No. 3869 (2014)
Open Access PDF DOI
Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting β1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.

Anna Ivanova, Yannis Kalaidzidis, Ronald Dirkx, Mihail Sarov, Michael Gerlach, Britta Schroth-Diez, Andreas Müller, Yanmei Liu, Cordula Andree, Bernard Mulligan, Carla Münster, Thomas Kurth, Marc Bickle, Stephan Speier, Konstantinos Anastassiadis, Michele Solimena
Age-dependent labeling and imaging of insulin secretory granules.
Diabetes, 62(11) 3687-3696 (2013)
Insulin is stored within the secretory granules of pancreatic β-cells, and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here, we illustrate a technology that enables the study of granule aging in insulinoma cells and β-cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic β-cells. Furthermore, the method is of general interest for the study of membrane traffic and aging.

Jerome Gilleron, William Querbes, Anja Zeigerer, Anna Borodovsky, Giovanni Marsico, Undine Schubert, Kevin Manygoats, Sarah Seifert, Cordula Andree, Martin Stöter, Hila Epstein-Barash, Ligang Zhang, Victor Koteliansky, Kevin Fitzgerald, Eugenio Fava, Marc Bickle, Yannis Kalaidzidis, Akin Akinc, Martin A Maier, Marino Zerial
Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.
Nat Biotechnol, 31(7) 638-646 (2013)
Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Athena Kyrkou, M Soufi, R Bahtz, Charles Ferguson, M Bai, Robert G. Parton, Ingrid Hoffmann, Marino Zerial, Theodore Fotsis, Carol Murphy
The RhoD to centrosomal duplication.
Small GTPases, 4(2) 116-122 (2013)
The main functional roles attributed to the centrosome, the major microtubule organizing center (MTOC) of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. In this commentary, we discuss our recent study, which shows the involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome's structural and functional integrity to vesicular trafficking.

A Kyrkou, M Soufi, R Bahtz, Charles Ferguson, M Bai, Robert G. Parton, I Hoffmann, Marino Zerial, Theodore Fotsis, C Murphy
RhoD participates in the regulation of cell-cycle progression and centrosome duplication.
Oncogene, 32(14) 1831-1842 (2013)
We have previously identified a Rho protein, RhoD, which localizes to the plasma membrane and the early endocytic compartment. Here, we show that a GTPase-deficient mutant of RhoD, RhoDG26V, causes hyperplasia and perturbed differentiation of the epidermis, when targeted to the skin of transgenic mice. In vitro, gain-of-function and loss-of-function approaches revealed that RhoD is involved in the regulation of G1/S-phase progression and causes overduplication of centrosomes. Centriole overduplication assays in aphidicolin-arrested p53-deficient U2OS cells, in which the cell and the centrosome cycles are uncoupled, revealed that the effects of RhoD and its mutants on centrosome duplication and cell cycle are independent. Enhancement of G1/S-phase progression was mediated via Diaph1, a novel effector of RhoD, which we have identified using a two-hybrid screen. These results indicate that RhoD participates in the regulation of cell-cycle progression and centrosome duplication.Oncogene advance online publication, 4 June 2012; doi:10.1038/onc.2012.195.

Enrico Perini
In vitro reconstitution of the molecular mechanisms of vesicle tethering and membrane fusion
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2013)

Varadharajan Sundaramurthy, Rico Barsacchi, Nikolay Samusik, Giovanni Marsico, Jerome Gilleron, Inna Kalaidzidis, Felix Meyenhofer, Marc Bickle, Yannis Kalaidzidis, Marino Zerial
Integration of chemical and RNAi multiparametric profiles identifies triggers of intracellular mycobacterial killing.
Cell Host Microbe, 13(2) 129-142 (2013)
Pharmacological modulators of host-microbial interactions can in principle be identified using high-content screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.

Jerome Gilleron, Anja Zeigerer, Giovanni Marsico, Thierry Galvez, Marino Zerial
[Key role of Rab5: from endosome biogenesis to liver metabolism].
Med Sci (Paris), 28(12) 1041-1044 (2012)

Thierry Galvez, Jerome Gilleron, Marino Zerial, Gregory A O'Sullivan
SnapShot: Mammalian Rab proteins in endocytic trafficking.
Cell, 151(1) Art. No. 234-234.e2. (2012)

Lionel Foret✳︎, Jonathan E Dawson✳︎, Roberto Villaseñor✳︎, Claudio Collinet, Andreas Deutsch, Lutz Brusch, Marino Zerial, Yannis Kalaidzidis, Frank Jülicher
A General Theoretical Framework to Infer Endosomal Network Dynamics from Quantitative Image Analysis.
Curr Biol, 22(15) 1381-1390 (2012)
BACKGROUND: Endocytosis allows the import and distribution of cargo into a series of endosomes with distinct morphological and biochemical characteristics. Our current understanding of endocytic cargo trafficking is based on the kinetics of net cargo transport between endosomal compartments without considering individual endosomes. However, endosomes form a dynamic network of membranes undergoing fusion and fission, thereby continuously exchanging and redistributing cargo. The macroscopic kinetic properties, i.e., the properties of the endosomal network as a whole, result from the collective behaviors of many individual endosomes, a problem so far largely unaddressed. RESULTS: Here, we developed a general theoretical framework to describe the dynamics of cargo distributions in the endosomal network. We combined the theory with quantitative experiments to study how the macroscopic kinetic properties of the endosomal network emerge from microscopic processes at the level of individual endosomes. We compared our theory predictions to experimental data in which dynamic distributions of endocytosed low-density lipoprotein (LDL) were quantified. CONCLUSIONS: Our theory can quantitatively describe the observed cargo distributions as a function of time. Remarkably, the theory allows determining microscopic kinetic parameters such as the fusion rate between endosomes from still images of cargo distributions at different times of internalization. We show that this method is robust and sensitive because cargo distributions result from an average over many stochastic events in many cells. Our results provide theoretical and experimental support to the "funnel model" of endosome progression and suggest that the conversion of early to late endosomes is the major mode of LDL trafficking.

David B Thompson, Roberto Villaseñor, Brent M Dorr, Marino Zerial, David R Liu
Cellular uptake mechanisms and endosomal trafficking of supercharged proteins.
Chem Biol, 19(7) 831-843 (2012)
Supercharged proteins (SCPs) can deliver functional macromolecules into the cytoplasm of mammalian cells more potently than unstructured cationic peptides. Thus far, neither the structural features of SCPs that determine their delivery effectiveness nor their intracellular fate postendocytosis, has been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge and that scGFPs enter cells through multiple pathways, including clathrin-dependent endocytosis and macropinocytosis. SCPs activate Rho and ERK1/2 and also alter the endocytosis of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of SCPs and implicate the modulation of endosomal trafficking as a determinant of macromolecule delivery efficiency.

Simone Fietz, Robert Lachmann, Holger Brandl, Martin Kircher, Nikolay Samusik, Roland Schröder, Naharajan Lakshmanaperumal, Ian Henry, Johannes Vogt, Axel Riehn, Wolfgang Distler, Robert Nitsch, Wolfgang Enard, Svante Pääbo, Wieland B. Huttner
Transcriptomes of germinal zones of human and mouse fetal neocortex suggest a role of extracellular matrix in progenitor self-renewal.
Proc Natl Acad Sci U.S.A., 109(29) 11836-11841 (2012)
The expansion of the neocortex during mammalian brain evolution results primarily from an increase in neural progenitor cell divisions in its two principal germinal zones during development, the ventricular zone (VZ) and the subventricular zone (SVZ). Using mRNA sequencing, we analyzed the transcriptomes of fetal human and embryonic mouse VZ, SVZ, and cortical plate. In mouse, the transcriptome of the SVZ was more similar to that of the cortical plate than that of the VZ, whereas in human the opposite was the case, with the inner and outer SVZ being highly related to each other despite their cytoarchitectonic differences. We describe sets of genes that are up- or down-regulated in each germinal zone. These data suggest that cell adhesion and cell-extracellular matrix interactions promote the proliferation and self-renewal of neural progenitors in the developing human neocortex. Notably, relevant extracellular matrix-associated genes include distinct sets of collagens, laminins, proteoglycans, and integrins, along with specific sets of growth factors and morphogens. Our data establish a basis for identifying novel cell-type markers and open up avenues to unravel the molecular basis of neocortex expansion during evolution.

C Schmees, Roberto Villaseñor, W Zheng, H Ma, Marino Zerial, Carl-Henrik Heldin, C Hellberg
Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V.
Mol Biol Cell, 23(13) 2571-2582 (2012)
Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

Julia Franziska Winter✳︎, Sebastian Höpfner✳︎, Kerstin Korn, Benjamin Farnung, Charles R. Bradshaw, Giovanni Marsico, Michael Volkmer, Bianca Habermann, Marino Zerial
Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity.
Nat Cell Biol, 14(7) 666-676 (2012)
Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.

Rico Barsacchi, Varadharajan Sundaramurthy, Kees Korbee, Jacques Neefjes, Tom H. M. Ottenhoff, Tiziana Scanu, Marino Zerial
Manipulating the Fight Between Human Host Cells and Intracellular Pathogens
In: Systems Microbiology: Current Topics and Applications. (Eds.) Brian D. Robertson, Brendan W. Wren,Norfolk,Caister Acad. Press (2012),77-94 Ch. 5

Anja Zeigerer, Jerome Gilleron, Roman L Bogorad, Giovanni Marsico, Hidenori Nonaka, Sarah Seifert, Hila Epstein-Barash, Satya Kuchimanchi, Chang Geng Peng, Vera M Ruda, Perla Del Conte-Zerial, Jan G Hengstler, Yannis Kalaidzidis, Victor Koteliansky, Marino Zerial
Rab5 is necessary for the biogenesis of the endolysosomal system in vivo.
Nature, 485(7399) 465-470 (2012)
An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Matthias Stein#, Manohar Pilli, Sabine Bernauer, Bianca Habermann, Marino Zerial, Rebecca C Wade#
The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.
PLoS ONE, 7(4) Art. No. e34870 (2012)
Open Access PDF DOI
Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.

Francisco Pan-Montojo, Mathias Schwarz, Christian Winkler, Mike Arnhold, Gregory A O'Sullivan, Arun Pal, Jonas Said, Giovanni Marsico, Jean-Marc Verbavatz, Margarita Rodrigo-Angulo, Gabriele Gille, Richard Funk, Heinz Reichmann
Environmental toxins trigger PD-like progression via increased alpha-synuclein release from enteric neurons in mice.
Sci Rep, 2 Art. No. 898 (2012)
Open Access PDF DOI
Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

Anton A Poznyakovskiy, Thomas Zahnert, Yannis Kalaidzidis, Nikoloz Lazurashvili, Rolf Schmidt, Hans-Jürgen Hardtke, Björn Fischer, Yury M Yarin
A segmentation method to obtain a complete geometry model of the hearing organ.
Hear Res, 282(1-2) 25-34 (2011)
We present a method for obtaining a complete geometry model of the fluid chambers of cochlea (scalae) from tomography images. An accurate segmentation of cochlea is problematic due to the low contrast of the inner membranes of scalae. Our method of 3D segmentation is based on dynamic resampling of an original image stack to achieve a perpendicular cross-section of the scalae on all sections. Subsequently, perpendicular cross-section is being segmented using 2D active contours. The center of mass of the contour is extracted and used to predict further course of scalae centerline by Kalman filter. Cross-section contours are subsequently assembled to the total geometry model. This method has been applied to CT images, but we expect that it could be used for segmentation of strongly curved low-contrast tubular objects recorded with other tomography techniques.

Asifa Akhtar, Elaine Fuchs, Timothy J. Mitchison, Reuben J Shaw, Daniel St Johnston, Andreas Strasser, Susan Taylor, Claire E. Walczak, Marino Zerial
A decade of molecular cell biology: achievements and challenges.
Nat Rev Mol Cell Biol, 12(10) 669-674 (2011)
Nature Reviews Molecular Cell Biology celebrated its 10-year anniversary during this past year with a series of specially commissioned articles. To complement this, here we have asked researchers from across the field for their insights into how molecular cell biology research has evolved during this past decade, the key concepts that have emerged and the most promising interfaces that have developed. Their comments highlight the broad impact that particular advances have had, some of the basic understanding that we still require, and the collaborative approaches that will be essential for driving the field forward.

Anna Urbanska, Lukasz Sadowski, Yannis Kalaidzidis, Marta Miaczynska
Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment
Traffic, 12(9) 1227-1241 (2011)
APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.

Iwona Pilecka, Lukasz Sadowski, Yannis Kalaidzidis, Marta Miaczynska
Recruitment of APPL1 to ubiquitin-rich aggresomes in response to proteasomal impairment
Exp Cell Res, 317(8) 1093-1107 (2011)
Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.

Giovanni Marsico
Analysis of the interplay between endocytosis and cell morphology through the development of computational methods
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2011)

Danming Tang, Yi Xiang, Stefano de Renzis, Jochen Rink, Gen Zheng, Marino Zerial, Yanzhuang Wang
The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle.
Nat Commun, 2 Art. No. 501 (2011)
Open Access PDF DOI
Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.

Akhila Chandrashaker
Systems analysis of early endosome motility through identification of molecular motors
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Akin Akinc, William Querbes, Soma De, June Qin, Maria Frank-Kamenetsky, K Narayanannair Jayaprakash, Muthusamy Jayaraman, Kallanthottathil G Rajeev, William L Cantley, J Robert Dorkin, James S Butler, Liuliang Qin, Timothy Racie, Andrew Sprague, Eugenio Fava, Anja Zeigerer, Michael J Hope, Marino Zerial, Dinah W Y Sah, Kevin Fitzgerald, Mark A Tracy, Muthiah Manoharan, Victor Koteliansky, Antonin de Fougerolles, Martin A Maier
Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.
Mol Ther, 18(7) 1357-1364 (2010)
Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Giovanna Mottola, Anne-Kathrin Classen, Marcos González-Gaitán, Suzanne Eaton, Marino Zerial
A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity.
Development, 137(14) 2353-2364 (2010)
In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. Here, we investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. We found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, further uncovered a novel function of this protein in PCP. At early stages of pupal wing development, the PCP protein Fmi redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. Our results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology.

Hesso Farhan, Markus W Wendeler, Sandra Mitrovic, Eugenio Fava, Yael Silberberg, Roded Sharan, Marino Zerial, Hans-Peter Hauri
MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening.
J Cell Biol, 189(6) 997-1011 (2010)
To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf-MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)-ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.

Dmitry Poteryaev✳︎, Sunando Datta✳︎, Karin Ackema, Marino Zerial, Anne Spang
Identification of the switch in early-to-late endosome transition.
Cell, 141(3) 497-508 (2010)
Sequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.

Claudio Collinet, Martin Stöter, Charles R. Bradshaw, Nikolay Samusik, Jochen Rink, Denise Kenski, Bianca Habermann, Frank Buchholz, Robert Henschel, Matthias S Mueller, Wolfgang E Nagel, Eugenio Fava, Yannis Kalaidzidis, Marino Zerial
Systems survey of endocytosis by multiparametric image analysis.
Nature, 464(7286) 243-249 (2010)
Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-beta and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

Livia Goto-Silva
Characterization of APPL signalling endosomes in neurons
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Dirk Helbing, Andreas Deutsch, Stefan Diez, Karsten Peters, Yannis Kalaidzidis, Kathrin Padberg-Gehle, Stefan Lämmer, Anders Johansson, Georg Breier, Frank Schulze, Marino Zerial
Biologistics and the struggle for efficiency: concepts and perspectives
Adv complex systems, 12(6) 533-548 (2009)
The growth of world population, limitation of resources, economic problems, and environmental issues force engineers to develop increasingly efficient solutions for logistic systems. Pure optimization for efficiency, however, has often led to technical solutions that are vulnerable to variations in supply and demand, and to perturbations. In contrast, nature already provides a large variety of efficient, flexible, and robust logistic solutions. Can we utilize biological principles to design systems, which can flexibly adapt to hardly predictable, fluctuating conditions? We propose a bio-inspired "BioLogistics" approach to deduce dynamic organization processes and principles of adaptive self-control from biological systems, and to transfer them to man-made logistics (including nanologistics), using principles of modularity, self-assembly, self-organization, and decentralized coordination. Conversely, logistic models can help revealing the logic of biological processes at the systems level.

Takeshi Ohya, Marta Miaczynska, Uenal Coskun, Barbara Lommer, Anja Runge, David N. Drechsel, Yannis Kalaidzidis, Marino Zerial
Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes.
Nature, 459(7250) 1091-1097 (2009)
Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.

Jesper B Bramsen, Maria B Laursen, Anne F Nielsen, Thomas B Hansen, Claus Bus, Niels Langkjaer, B Ravindra Babu, Torben Højland, Mikhail Abramov, Arthur Van Aerschot, Dalibor Odadzic, Romualdas Smicius, Jens Haas, Cordula Andree, Jharna Barman, Malgorzata Wenska, Puneet Srivastava, Chuanzheng Zhou, Dmytro Honcharenko, Simone Hess, Elke Müller, Georgii V Bobkov, Sergey N Mikhailov, Eugenio Fava, Thomas F Meyer, Jyoti Chattopadhyaya, Marino Zerial, Joachim W Engels, Piet Herdewijn, Jesper Wengel, Jørgen Kjems
A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.
Nucleic Acids Res, 37(9) 2867-2881 (2009)
Open Access PDF DOI
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Claudio Collinet
System survey of endocytosis by functional genomics and quantitative multi-parametric image analysis
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2009)

Yonathan Lissanu Deribe, Philipp Wild, Akhila Chandrashaker, Jasna Curak, Mario Schmidt, Yannis Kalaidzidis, Natasa Milutinovic, Irina Kratchmarova, Lukas Buerkle, Michael J Fetchko, Philipp Schmidt, Saranya Kittanakom, Kevin R Brown, Igor Jurisica, Blagoy Blagoev, Marino Zerial, Igor Stagljar, Ivan Dikic
Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6.
Sci Signal, 2(102) Art. No. ra84 (2009)
Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.

Yannis Kalaidzidis
Multiple objects tracking in fluorescence microscopy.
J Math Biol, 58(1-2) 57-80 (2009)
Many processes in cell biology are connected to the movement of compact entities: intracellular vesicles and even single molecules. The tracking of individual objects is important for understanding cellular dynamics. Here we describe the tracking algorithms which have been developed in the non-biological fields and successfully applied to object detection and tracking in biological applications. The characteristics features of the different algorithms are compared.

Charles R. Bradshaw
Improving the analysis of genome-wide screens through the detection of weakly conserved domains
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2009)

Anton A Poznyakovskiy, Thomas Zahnert, Yannis Kalaidzidis, Rolf Schmidt, Björn Fischer, Johannes Baumgart, Yury M Yarin
The creation of geometric three-dimensional models of the inner ear based on micro computer tomography data.
Hear Res, 243(1-2) 95-104 (2008)
The modeling of the mechanical process of hearing requires an accurate geometrical model of the inner ear (cochlea). The purpose of this study was the creation of a 3-D model of the fluid chambers of Guinea pig cochlea, which could serve as a basis for further mechanical modeling. Micro computer tomography used in this study is a noninvasive method to visualize bony structures. The visualization of the membranous labyrinth was achieved by additional staining of the specimen with OsO(4). The resulting stack of images has been transformed into a cylindrical coordinate system. To suppress noise on tomography images, a nonlinear smoothing method, anisotropic diffusion, were applied. A new approach has been proposed to estimate algorithm parameters automatically. Then, a segmentation using active contours (snakes) was performed. In this study, a new energy linking the contours on adjacent slices has been added to the standard approach. This compensates the inconsistencies between adjacent contours. The images segmented in this way were used as a basis for a 3-D reconstruction of the hearing organ.

Marisa P. McShane, Marino Zerial
Survival of the weakest: signaling aided by endosomes.
J Cell Biol, 182(5) 823-825 (2008)
The tyrosine kinase receptor c-Met plays a key role in cell proliferation, morphogenesis, and motility in response to hepatocyte growth factor. C-Met is often altered in cancer and is a major target for therapeutic intervention. Despite knowing a great deal of the molecular machinery downstream of this receptor tyrosine kinase, the spatiotemporal regulation of c-Met signaling still remains elusive. In this issue of the Journal of Cell Biology, Kermorgant and Parker (Kermorgant, S. and P.J. Parker. 2008. J. Cell Biol. 182:855-863) provide evidence for a model in which the c-Met-activated STAT3 signal is mediated by endosomal trafficking. This study elegantly highlights how weak signals can be effectively transmitted to the nucleus by exploiting endosomal compartments, raising important mechanistic implications for the signaling research community.

Annette Schenck, Livia Goto-Silva, Claudio Collinet, Muriel Rhinn, Angelika Giner, Bianca Habermann, Michael Brand, Marino Zerial
The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development.
Cell, 133(3) 486-497 (2008)
During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

Arun Pal, Fedor F. Severin, Sebastian Höpfner, Marino Zerial
Regulation of endosome dynamics by Rab5 and Huntingtin-HAP40 effector complex in physiological versus pathological conditions
In: Small GTPases in disease. (Eds.) William E. Balch Methods in enzymology ; 438.,Amsterdam, Netherlands,Elsevier (2008),239-257 Ch. 17
Vesicular transport of signaling molecules, specifically neurotrophins, in neurons is essential for their differentiation, survival, and plasticity. Neurotrophins such as neuron growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are internalized by receptor-mediated endocytosis at synaptic terminals and loaded into endosomes for microtubule-based transport along axons to the cell body where they exert their signaling function in the nucleus. The molecular mechanisms underlying this intracellular transport are not only relevant from a basic knowledge viewpoint, but have also important implications for neurodegenerative diseases. Defects in trafficking are increasingly implicated in the pathology of Huntington's disease (HD) and other neurodegenerative disorders. The small GTPases Rab5 and Rab7 play important roles in the endocytic trafficking of neurotrophins. We have recently identified Huntingtin (Htt) and Huntingtin associated protein of 40 kDa (HAP40) as a novel Rab5 effector complex that regulates endosome motility. In HD, we detected higher HAP40 protein levels compared with normal cells. Such increase causes an augmented recruitment of Htt onto Rab5-positive early endosomes that drastically reduces their motility by "switching" these organelles from microtubules to F-actin. These findings suggest a mechanism by which impaired Rab5-mediated trafficking of neurotrophic factors may be a key event of the pathogenetic process leading to neurodegeneration in HD. To dissect the mechanisms by which Htt, HAP40, and Rab5 function in early endosome interactions with the cytoskeleton, we developed assays to investigate endosome-cytoskeleton interactions that can be applied to normal and pathological conditions. We provide here detailed protocols for, first, an assay that measures binding of early endosomes to microtubules and F-actin. Second, we describe an improved protocol for a cell-free assay that recapitulates the motility of early endosomes along microtubules in vitro. These assays provide mechanistic insights into the dysfunction of endosome motility occurring in HD as well as other neurodegenerative disorders.

Perla Del Conte-Zerial✳︎, Lutz Brusch✳︎, Jochen Rink, Claudio Collinet, Yannis Kalaidzidis, Marino Zerial, Andreas Deutsch
Membrane identity and GTPase cascades regulated by toggle and cut-out switches.
Mol Syst Biol, 4 206-206 (2008)
Open Access PDF DOI
Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5-to-Rab7 conversion is rather based on a newly characterized 'cut-out switch' analogous to an electrical safety-breaker. Both designs require cooperativity of auto-activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut-out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut-out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes.

Julia Franziska Winter
Genome-wide analysis of membrane trafficking in polarised epithelial cells in the C. elegans intestine
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2008)

Yannis Kalaidzidis
Intracellular objects tracking.
Eur J Cell Biol, 86(9) 569-578 (2007)
The tracking of intracellular organelles and vesicles is becoming increasingly important for understanding cellular dynamics. Originally, the development of tracking algorithms was mainly pursued in other fields, e.g. aerospace/military/street surveillance. However, most of this algorithm is not directly applicable to live cell microscopy data. Here we describe the algorithms that have been successfully applied to object detection and tracking specifically in vivo and in vitro motility assays. The characteristics advantages and disadvantages of the different approaches are compared.

Torben Lessmann, Michele G Leuenberger, Sascha Menninger, Meritxell Lopez-Canet, Oliver Müller, Stefan Hümmer, Jenny Bormann, Kerstin Korn, Eugenio Fava, Marino Zerial, Thomas U Mayer, Herbert Waldmann
Natural product-derived modulators of cell cycle progression and viral entry by enantioselective oxa Diels-Alder reactions on the solid phase.
Chem Biol, 14(4) 443-451 (2007)
The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired alpha,beta-unsaturated delta-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity.

Anna Kicheva✳︎, Periklis Pantazis✳︎, Tobias Bollenbach✳︎, Yannis Kalaidzidis, Thomas Bittig, Frank Jülicher#, Marcos González-Gaitán#
Kinetics of morphogen gradient formation.
Science, 315(5811) 521-525 (2007)
In the developing fly wing, secreted morphogens such as Decapentaplegic (Dpp) and Wingless (Wg) form gradients of concentration providing positional information. Dpp forms a longer-range gradient than Wg. To understand how the range is controlled, we measured the four key kinetic parameters governing morphogen spreading: the production rate, the effective diffusion coefficient, the degradation rate, and the immobile fraction. The four parameters had different values for Dpp versus Wg. In addition, Dynamin-dependent endocytosis was required for spreading of Dpp, but not Wg. Thus, the cellular mechanisms of Dpp and Wingless spreading are different: Dpp spreading requires endocytic, intracellular trafficking.

Arun Pal, Fedor F. Severin, Barbara Lommer, Anna Shevchenko, Marino Zerial
Huntingtin-HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease.
J Cell Biol, 172(4) 605-618 (2006)
The molecular mechanisms underlying the targeting of Huntingtin (Htt) to endosomes and its multifaceted role in endocytosis are poorly understood. In this study, we have identified Htt-associated protein 40 (HAP40) as a novel effector of the small guanosine triphosphatase Rab5, a key regulator of endocytosis. HAP40 mediates the recruitment of Htt by Rab5 onto early endosomes. HAP40 overexpression caused a drastic reduction of early endosomal motility through their displacement from microtubules and preferential association with actin filaments. Remarkably, endogenous HAP40 was up-regulated in fibroblasts and brain tissue from human patients affected by Huntington's disease (HD) as well as in STHdhQ(111) striatal cells established from a HD mouse model. These cells consistently displayed altered endosome motility and endocytic activity, which was restored by the ablation of HAP40. In revealing an unexpected link between Rab5, HAP40, and Htt, we uncovered a new mechanism regulating cytoskeleton-dependent endosome dynamics and its dysfunction under pathological conditions.

Sally Martin, Kim Driessen, Susan J. Nixon, Marino Zerial, Robert G. Parton
Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism.
J Biol Chem, 280(51) 42325-42335 (2005)
Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Jochen Rink, Eric Ghigo, Yannis Kalaidzidis, Marino Zerial
Rab conversion as a mechanism of progression from early to late endosomes.
Cell, 122(5) 735-749 (2005)
The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.

Gaëtane Macé, Marta Miaczynska, Marino Zerial, Angel R Nebreda
Phosphorylation of EEA1 by p38 MAP kinase regulates mu opioid receptor endocytosis.
EMBO J, 24(18) 3235-3246 (2005)
Morphine analgesic properties and side effects such as tolerance are mediated by the mu opioid receptor (MOR) whose endocytosis is considered of primary importance for opioid pharmacological effects. Here, we show that p38 mitogen-activated protein kinase (MAPK) activation is required for MOR endocytosis and sufficient to trigger its constitutive internalization in the absence of agonist. Further studies established a functional link between p38 MAPK and the small GTPase Rab5, a key regulator of endocytosis. Expression of an activated mutant of Rab5 stimulated endocytosis of MOR ligand-independently in wild-type but not in p38alpha-/- cells. We found that p38alpha can phosphorylate the Rab5 effectors EEA1 and Rabenosyn-5 on Thr-1392 and Ser-215, respectively, and these phosphorylation events regulate the recruitment of EEA1 and Rabenosyn-5 to membranes. Moreover, phosphomimetic mutation of Thr-1392 in EEA1 can bypass the requirement for p38alpha in MOR endocytosis. Our results highlight a novel mechanism whereby p38 MAPK regulates receptor endocytosis under physiological conditions via phosphorylation of Rab5 effectors.

Hye-Won Shin, Mitsuko Hayashi, Savvas Christoforidis, Sandra Lacas-Gervais, Sebastian Hoepfner, Markus R Wenk, Jan Modregger, Sandrine Uttenweiler-Joseph, Matthias Wilm, Arne Nystuen, Wayne N Frankel, Michele Solimena, Pietro De Camilli, Marino Zerial
An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway.
J Cell Biol, 170(4) 607-618 (2005)
Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Lucas Pelkmans, Marino Zerial
Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae.
Nature, 436(7047) 128-133 (2005)
A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one 'quantum') of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.

Lucas Pelkmans, Eugenio Fava, Hannes Grabner, Michael Hannus, Bianca Habermann, Eberhard Krausz, Marino Zerial
Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis.
Nature, 436(7047) 78-86 (2005)
Endocytosis is a key cellular process, encompassing different entry routes and endocytic compartments. To what extent endocytosis is subjected to high-order regulation by the cellular signalling machinery remains unclear. Using high-throughput RNA interference and automated image analysis, we explored the function of human kinases in two principal types of endocytosis: clathrin- and caveolae/raft-mediated endocytosis. We monitored this through infection of vesicular stomatitis virus, simian virus 40 and transferrin trafficking, and also through cell proliferation and apoptosis assays. Here we show that a high number of kinases are involved in endocytosis, and that each endocytic route is regulated by a specific kinase subset. Notably, one group of kinases exerted opposite effects on the two endocytic routes, suggesting coordinate regulation. Our analysis demonstrates that signalling functions such as those controlling cell adhesion, growth and proliferation, are built into the machinery of endocytosis to a much higher degree than previously recognized.

Sebastian Höpfner
Modulation of Cargo Transport and Sorting through Endosome Motility and Positioning
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2005)

Sebastian Hoepfner, Fedor F. Severin, Alicia Cabezas, Bianca Habermann, Anja Runge, David J. Gillooly, Harald Stenmark, Marino Zerial
Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B.
Cell, 121(3) 437-450 (2005)
Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.

Eberhard Krausz, Lucas Pelkmans, Eugenio Fava, Michael Hannus, Claudia Möbius, Kerstin Korn, Francoise Halley, Annett Lohmann, Hannes Grabner, Marino Zerial
Genomweite funktionelle Analyse des Kinoms mittels RNA-Interfernz
BIOspektrum, 11(Sonderausgabe) 518-520 (2005)
In den Laboratorien des „High-Throughput Technology Development Studio“ (TDS) werden in enger Zusammenarbeit mit internen und externen Forschungsgruppen phänotypische Assays (qualitative/ quantitative Untersuchungen) entwickelt und mikroskopiegestützte Durchmusterungen, so genanntes „High-Content Screening“, sowohl in kultivierten Zellen als auch in Modellorganismen durchgeführt. Dabei werden automatisierte Pipettierstationen, Mikroskope und Bildverarbeitungsprogramme zu Hilfe genommen, um neue Genfunktionen oder Wirkungsweisen chemischer Substanzen zu entdecken.

Carsten Schnatwinkel, Savvas Christoforidis, Margaret R Lindsay, Sandrine Uttenweiler-Joseph, Matthias Wilm, Robert G. Parton, Marino Zerial
The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.
PLoS Biol, 2(9) 261-261 (2004)
Open Access PDF DOI
The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

Lucas Pelkmans, Thomas Bürli, Marino Zerial, Ari Helenius
Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic.
Cell, 118(6) 767-780 (2004)
Endocytosis comprises several routes of internalization. An outstanding question is whether the caveolar and endosomal pathways intersect. Following transport of the caveolar protein Caveolin-1 and two cargo complexes, Simian Virus 40 and Cholera toxin, in live cells, we uncovered a Rab5-dependent pathway in which caveolar vesicles are targeted to early endosomes and form distinct and stable membrane domains. In endosomes, the low pH selectively allowed the toxin to diffuse out of the caveolar domains into the surrounding membrane, while the virus remained trapped. Thus, we conclude that, unlike cyclic assembly and disassembly of coat proteins in vesicular transport, oligomeric complexes of caveolin-1 confer permanent structural stability to caveolar vesicles that transiently interact with endosomes to form subdomains and release cargo selectively by compartment-specific cues.

Marta Miaczynska, Lucas Pelkmans, Marino Zerial
Not just a sink: endosomes in control of signal transduction.
Curr Opin Cell Biol, 16(4) 400-406 (2004)
Recent studies indicate that endocytic organelles can play a more active role in signal propagation and amplification than was recognised before. By deciphering the interplay between endocytosis and signalling, we will be able to gain a more sophisticated level of understanding of signal transduction mechanisms.

Marta Miaczynska, Savvas Christoforidis, Angelika Giner, Anna Shevchenko, Sandrine Uttenweiler-Joseph, Bianca Habermann, Matthias Wilm, Robert G. Parton, Marino Zerial
APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment.
Cell, 116(3) 445-456 (2004)
Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Stéphane Gasman, Yannis Kalaidzidis, Marino Zerial
RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase.
Nat Cell Biol, 5(3) 195-204 (2003)
Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.

Rafael Mattera, Cecilia N Arighi, Robert Lodge, Marino Zerial, Juan S Bonifacino
Divalent interaction of the GGAs with the Rabaptin-5-Rabex-5 complex.
EMBO J, 22(1) 78-88 (2003)
Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5-Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439-443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the gamma1- and gamma2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA-Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)-Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gamma-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

James T Murray, Christina Panaretou, Harald Stenmark, Marta Miaczynska, Jonathan M Backer
Role of Rab5 in the recruitment of hVps34/p150 to the early endosome.
Traffic, 3(6) 416-427 (2002)
PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo. We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.

Ekaterini Panopoulou, David J. Gillooly, Jeffrey L Wrana, Marino Zerial, Harald Stenmark, Carol Murphy, Theodore Fotsis
Early endosomal regulation of Smad-dependent signaling in endothelial cells.
J Biol Chem, 277(20) 18046-18052 (2002)
Transforming growth factor beta (TGFbeta) receptors require SARA for phosphorylation of the downstream transducing Smad proteins. SARA, a FYVE finger protein, binds to membrane lipids suggesting that activated receptors may interact with downstream signaling molecules at discrete endocytic locations. In the present study, we reveal a critical role for the early endocytic compartment in regulating Smad-dependent signaling. Not only is SARA localized on early endosomes, but also its minimal FYVE finger sequence is sufficient for early endosomal targeting. Expression of a SARA mutant protein lacking the FYVE finger inhibits downstream activin A signaling in endothelial cells. Moreover, a dominant-negative mutant of Rab5, a crucial protein for early endosome dynamics, causes phosphorylation and nuclear translocation of Smads leading to constitutive (i.e. ligand independent) transcriptional activation of a Smad-dependent promoter in endothelial cells. As inhibition of endocytosis using the K44A negative mutant of dynamin and RN-tre did not lead to activation of Smad-dependent transcription, the effects of the dominant-negative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGFbeta/activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGFbeta/activin signal transduction pathways.

Stefano de Renzis, Birte Sönnichsen, Marino Zerial
Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes.
Nat Cell Biol, 4(2) 124-133 (2002)
The three GTPases Rab5, Rab4 and Rab11 regulate sequential transport steps along the endocytic/recycling pathway, and occupy distinct membrane domains on early and recycling endosomes. To address the mechanisms that regulate communication between such domains, we searched for proteins that interact with both Rab5 and Rab4. Here, we report that Rabenosyn-5, a previously identified Rab5 effector, also binds to Rab4. Rabenosyn-5 overexpression increased the association between Rab5 and Rab4 endosomal domains and decreased the fraction of Rab4- and Rab11-positive structures. This redistribution was accompanied by a faster rate of transferrin recycling from early endosomes to the cell surface and reduced transport to Rab11-containing perinuclear recycling endosomes. These effects depend on the ability of Rabenosyn-5 to interact with Rab4. We propose that divalent Rab effectors regulate protein sorting and recycling by connecting Rab domains on early endosomes.

Marta Miaczynska, Marino Zerial
Mosaic organization of the endocytic pathway.
Exp Cell Res, 272(1) 8-14 (2002)

Roger Lippé, Marta Miaczynska, Vladimir Rybin, Anja Runge, Marino Zerial
Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex.
Mol Biol Cell, 12(7) 2219-2228 (2001)
Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

Heike Esters, Kirill Alexandrov, Andrei Iakovenko, Tania Ivanova, Nicolas Thomä, Vladimir Rybin, Marino Zerial, Axel J. Scheidig, Roger S. Goody
Vps9, Rabex-5 and DSS4: proteins with weak but distinct nucleotide-exchange activities for Rab proteins.
J Mol Biol, 310(1) 141-156 (2001)
The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.

Carol Murphy, Rainer Saffrich, Jean-Christophe Olivo-Marin, Angelika Giner, Wilhelm Ansorge, Theodore Fotsis, Marino Zerial
Dual function of rhoD in vesicular movement and cell motility.
Eur J Cell Biol, 80(6) 391-398 (2001)
The trafficking of intracellular membranes requires the coordination of membrane-cytoskeletal interactions. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions. We have previously identified a rho protein, rhoD, which is localized to the plasma membrane and early endosomes. When overexpressed, rhoD alters the actin cytoskeleton and plays an important role in endosome organization. We found that a rhoD mutant exerts its effect on early endosome dynamics through an inhibition in organelle motility. In these studies, the effect of rhoD on endosome dynamics was evaluated in the presence of a constitutively active, GTPase-deficient mutant of rab5, rab5Q79L. As rab5Q79L itself stimulates endosome motility, rhoD might counteract this stimulation, without itself exerting any effect in the absence of rab5 activation. We have now addressed this issue by investigating the effect of rhoD in the absence of co-expressed rab5. We find that rhoDG26V alone alters vesicular dynamics. Vesicular movement, in particular the endocytic/recycling circuit, is altered during processes such as cell motility. Due to the participation of vesicular motility and cytoskeletal rearrangements in cell movement and the involvement of rhoD in both, we have addressed the role of rhoD in this process and have found that rhoDG26V inhibits endothelial cell motility.

Sandrine Uttenweiler-Joseph, Gitte Neubauer, Savvas Christoforidis, Marino Zerial, Matthias Wilm
Automated de novo sequencing of proteins using the differential scanning technique.
Proteomics, 1(5) 668-682 (2001)
Despite the progress in genomic DNA sequencing de novo sequencing of peptides is still required in a biological research environment since many experiments are done in organisms whose genomes are not sequenced. A way to unambiguously retrieve a peptide sequence from a tandem mass spectrum is to assign the correct ion type to the fragments. Here we describe a method which improves the specificity in y-ion assignment throughout the spectrum. The differential scanning technique requires that the peptides are partially 18O labelled at their C-terminus and that two fragment spectra are acquired for each peptide, one selecting the 16O/18O isotopic cluster and a second fragmenting only the 18O labelled ions. When the spectra are acquired with a quadrupole time of flight mass spectrometer y-ions can be very specifically filtered from the spectrum using a computer algorithm. Partial or complete peptide sequences can be assigned automatically simply by finding the most abundant series of fragments spaced by amino acid residue masses. This method was used extensively in a project investigating vesicular transport in bovine brain cells. Human or mouse homologues to the bovine proteins were found in EST databases facilitating rapid cloning of the human homologues.

Tom Van de Putte, An Zwijsen, Olivier Lonnoy, Vladimir Rybin, Miranda Cozijnsen, Annick Francis, Veerle Baekelandt, Christine A. Kozak, Marino Zerial, Danny Huylebroeck
Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.
Mech Dev, 102(1-2) 33-44 (2001)
In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

Marino Zerial, Heidi M. McBride
Rab proteins as membrane organizers.
Nat Rev Mol Cell Biol, 2(2) 107-117 (2001)
Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport. Rab proteins and their effectors coordinate consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles to their target compartment. These molecules are highly compartmentalized in organelle membranes, making them excellent candidates for determining transport specificity and organelle identity.

Savvas Christoforidis, Marino Zerial
Purification of EEA1 from bovine brain cytosol using Rab5 affinity chromatography and activity assays
In: GTPases involved in vesicular traffic. (Eds.) William E. Balch Methods in enzymology ; 329.,Amsterdam, Netherlands,Academic Press (2001),120-132 Ch. 14

Harald Stenmark, Marino Zerial
Molecular mechanisms of membrane fusion in the endocytic pathway
In: Endocytosis. (Eds.) Mark Marsh Frontiers in molecular biology ; 36.,Oxford, UK,Oxford University Press (2001),94-110 Ch. 5

Erik Nielsen, Fedor F. Severin, Anthony A. Hyman, Marino Zerial
In vitro reconstitution of endosome motility along microtubules
In: Kinesin protocols. (Eds.) Isabelle Vernos Methods in molecular biology ; 164.,Totowa, USA,Humana Press (2001),133-146 Ch. 12

Roger Lippé, Hisanori Horiuchi, Anja Runge, Marino Zerial
Expression, purification, and characterization of Rab5 effector complex, rabaptin-5/rabex-5
In: GTPases involved in vesicular traffic. (Eds.) William E. Balch Methods in enzymology ; 329.,Amsterdam, Netherlands,Elsevier (2001),132-145

Letizia Lanzetti, Vladimir Rybin, Maria Grazia Malabarba, Savvas Christoforidis, Giorgio Scita, Marino Zerial, Pier Paolo Di Fiore
The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5.
Nature, 408(6810) 374-377 (2000)
How epidermal growth factor receptor (EGFR) signalling is linked to EGFR trafficking is largely unknown. Signalling and trafficking involve small GTPases of the Rho and Rab families, respectively. But it remains unknown whether the signalling relying on these two classes of GTPases is integrated, and, if it is, what molecular machinery is involved. Here we report that the protein Eps8 connects these signalling pathways. Eps8 is a substrate of the EGFR, which is held in a complex with Sos1 by the adaptor protein E3bl (ref. 2), thereby mediating activation of Rac. Through its src homology-3 domain, Eps8 interacts with RN-tre. We show that RN-tre is a Rab5 GTPase-activating protein, whose activity is regulated by the EGFR. By entering in a complex with Eps8, RN-tre acts on Rab5 and inhibits internalization of the EGFR. Furthermore, RN-tre diverts Eps8 from its Rac-activating function, resulting in the attenuation of Rac signalling. Thus, depending on its state of association with E3b1 or RN-tre, Eps8 participates in both EGFR signalling through Rac, and trafficking through Rab5.

Erik Nielsen, Savvas Christoforidis, Sandrine Uttenweiler-Joseph, Marta Miaczynska, Frederique Dewitte, Matthias Wilm, Bernard Hoflack, Marino Zerial
Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain.
J Cell Biol, 151(3) 601-612 (2000)
Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

Sanna Lehtonen, Ari Ora, Vesa M Olkkonen, Lin Geng, Marino Zerial, Stefan Somlo, Eero Lehtonen
In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
J Biol Chem, 275(42) 32888-32893 (2000)
We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Birte Sönnichsen, Stefano de Renzis, Erik Nielsen, Jens Rietdorf, Marino Zerial
Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.
J Cell Biol, 149(4) 901-914 (2000)
Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

Savvas Christoforidis, Marino Zerial
Purification and identification of novel Rab effectors using affinity chromatography.
Methods, 20(4) 403-410 (2000)
Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.

Mariantonietta Rubino, Marta Miaczynska, Roger Lippé, Marino Zerial
Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes.
J Biol Chem, 275(6) 3745-3748 (2000)
The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.

Savvas Christoforidis, Marta Miaczynska, Keith Ashman, Matthias Wilm, Liyun Zhao, Shu-Chin Yip, Michael D. Waterfield, Jonathan M Backer, Marino Zerial
Phosphatidylinositol-3-OH kinases are Rab5 effectors.
Nat Cell Biol, 1(4) 249-252 (1999)

Heidi M. McBride, Vladimir Rybin, Carol Murphy, Angelika Giner, Rohan Teasdale, Marino Zerial
Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.
Cell, 98(3) 377-386 (1999)
SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.

Jon Mulholland, James Konopka, Birgit Singer-Kruger, Marino Zerial, David Botstein
Visualization of receptor-mediated endocytosis in yeast.
Mol Biol Cell, 10(3) 799-817 (1999)
We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.

Savvas Christoforidis, Heidi M. McBride, Robert D. Burgoyne, Marino Zerial
The Rab5 effector EEA1 is a core component of endosome docking.
Nature, 397(6720) 621-625 (1999)
Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5. Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

Sanna Lehtonen, Vesa M Olkkonen, Mark Stapleton, Marino Zerial, Eero Lehtonen
HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis.
Int J Dev Biol, 42(6) 775-782 (1998)
We used the differential hybridization technique for isolating developmentally regulated genes from the mouse metanephric kidney. In this screening, we identified the cDNA encoding high-mobility-group protein 17 (HMG-17), a chromosomal non-histone protein which modulates the conformation of transcriptionally active chromatin. Using Northern blot analysis, the HMG-17 mRNA was strongly expressed during embryogenesis and downregulated in various adult murine organs. At the histological level, the transcript localized to differentiating tissue regions and was apparently downregulated in mature structures indicating that HMG-17 expression is linked to cell differentiation. HMG-17 can thus be regarded as a general marker for tissues or cells undergoing differentiation during organogenesis.

Anne Simonsen✳︎, Roger Lippé✳︎, Savvas Christoforidis, Jean-Michel Gaullier, Andreas Brech, Judy Callaghan, Ban-Hock Toh, Carol Murphy, Marino Zerial, Harald Stenmark
EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.
Nature, 394(6692) 494-498 (1998)
GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Helene Gournier, Harald Stenmark, Vladimir Rybin, Roger Lippé, Marino Zerial
Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.
EMBO J, 17(7) 1930-1940 (1998)
Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Gaetano Vitale, Vladimir Rybin, Savvas Christoforidis, Per-Ove Thornqvist, Mary McCaffrey, Harald Stenmark, Marino Zerial
Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.
EMBO J, 17(7) 1941-1951 (1998)
Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

Paola Zacchi, Harald Stenmark, Robert G. Parton, Donata Orioli, Filip Lim, Angelika Giner, Ira Mellman, Marino Zerial, Carol Murphy
Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells.
J Cell Biol, 140(5) 1039-1053 (1998)
A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Harald Stenmark, Marino Zerial
Transient Expression Using the T7 RNA Polymerase Recombinant Vaccinia Virus System
In: Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 4. (Eds.) Julio E. Celis,Amsterdam, Netherlands,Academic Press (1998),201-203 Ch. 14

Harald Stenmark, Marino Zerial
Stock Preparation of Recombinant Vaccinia Virus
In: Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 1. (Eds.) Julio E. Celis,Amsterdam, Netherlands,Academic Press (1998),540-543
Recombinant vaccinia virus is useful for the transient expression of exogenous proteins in a variety of cell types. This article describes a simple method for preparing large stocks of the virus. The virus is very stable at -80C, and once a virus stock has been made, it can be used for experiments for several years, if required.

Marino Zerial
Membranes and sorting
Curr Opin Cell Biol, 10 475-476 (1998)

Harald Stenmark, Marino Zerial
In: Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 4. (Eds.) Julio E. Celis,Amsterdam, Netherlands,Academic Press (1997),141-144 Ch. 13