Principles of cell and tissue organization: from endocytosis to a systems understanding of liver structure and function

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Multi-Scale Analysis of Cell and Tissue Organization

The Zerial group works on endocytosis - from its molecular mechanisms to implications in cell and tissue organization. Our interests range from the assembly and functional characterization of the endosomal fusion machinery to the establishment of hepatocyte cell polarity and liver tissue structure and function. Our group uses a unique interdisciplinary approach combining biochemical and biophysical methods with advanced light microscopy, cell biology and computer-aided three-dimensional tissue reconstruction.

Functional Characterization of the Endosomal Fusion Machinery

Our research group pioneered the functional analysis of the Rab family of small GTPases in intracellular transport with a special focus on Rab5. Rab5 and its effector proteins play a key role in endosomal membrane fusion. By reconstituting an endosomal tethering machinery in vitro, we uncovered a new mechanism in which Rab5 induces a change in flexibility of the membrane tether EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane leading to membrane fusion. We also succeeded in reconstituting a synthetic endosome that recapitulates the functional properties of the cellular organelle. Now, we aim at elucidating the molecular mechanism at each step of the process.

From Molecular Scale to Cell and Tissue Organization and Function

In recent years, our research interest broadened from molecular mechanisms to cell and tissue function. We work mainly on mouse liver as a model organ because of its extraordinary features of tissue organization and cell polarization and regeneration potential. Currently, we are especially interested in the role of the endosomal pathway in cell polarity establishment and tissue formation. We are furthermore exploring the mechanisms of tissue regeneration and organization.

In our research, we use an interdisciplinary approach combining state-of-the-art microscopy techniques and development of advanced computer-aided 3D tissue reconstruction algorithms to understand the structure-function relationship of liver tissue. Using this approach, we advanced the understanding of liver tissue organization and predicted alterations occurring in liver disease.

Our long-term goal is to develop a theory of liver tissue organization integrating different scales - from the molecular to the organ scale - and use it to predict tissue structure and function in health and disease.

Exploiting the Endosomal System for Delivery of Macromolecules

In medicine, the use of biologically active macromolecules as therapeutics is becoming a widely used strategy. Cells take up macromolecules via the endocytic pathway and traffic them to their specific site of action within the cell. Currently, uptake and endosomal escape of the molecule from vesicles into the cytoplasm are still poorly understood and challenging steps in drug development and delivery. Therefore, our lab has been exploring uptake mechanisms and endosomal escape in various collaborations with pharmaceutical companies. Our expertise in endosomal trafficking and membrane fusion allows us to use bio-inspired approaches to improve macromolecule delivery systems.

Selected publications

* joined first author # joined corresponding author

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.

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.

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.

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.

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