Stochastic phenomena play a fundamental role in many biological systems, ranging from gene regulation to cell-fate determination. Understanding such phenomena raises new theoretical challenges at the interface between stochastic processes, statistical physics and computation. In this talk I will present some of our group’s work in this direction. In the first part of the talk, I will show how cells can use phase coexistence to control and suppress protein concentration fluctuations. Using a non-equilibrium model that links active protein synthesis and turnover to the physics of phase separation, I will show that concentration fluctuations can be strongly reduced in the presence of phase separated compartments. I will present experimental single-cell data in synthetic and endogenous compartments, which support this prediction. In the second part of my talk, I will focus on inverse problems and how stochastic processes can be robustly inferred from limited experimental data. As an example, I will present a statistical method to quantify CTCF/cohesion-mediated chromatin looping dynamics from two-point live-imaging measurements. The method combines a simple polymer model with a Bayesian filtering approach to infer loop lifetimes and frequencies. When applied to experimental data, this method revealed that chromatin loops are surprisingly rare and short-lived. I will conclude my talk by outlining several important challenges for the future.

We are interested in the reciprocal interactions between form and function. To explore how physiology interacts with cell and tissue-wide processes, we follow a wide range of approaches using zebrafish, mice, and physical models. Investigating the morphogenesis of the notochord we found that its relatively simple architecture allows us to explore biological processes including periodic patterning, axial mechanics, and volume control. These processes offer inroads into long-range tissue-wide interactions and self-organization.

About 10% of couples are unintentionally childless. The causes of infertility often lie in the egg. As women age, more and more eggs become defective. Melina Schuh, Director at the Max Planck Institute for Multidisciplinary Sciences, studies how eggs develop, how they function and why they are so often defective. In her presentation, she will talk about how eggs support embryo development and about new methods that could help more couples to conceive.

Macrophages are the earliest set of immune cells that populate the murine skin. However, much less is understood about their role in regula;ng developmental and inflammatory programs in the developing skin. Our study suggests that macrophages in the developing skin can respond to sterile inflammatory cues by augmen;ng the synthesis of ECM (extracellular matrix) remodeling enzymes called MMPs (matrix metalloproteinases). These MMPs exacerbate chronic inflamma;on by genera;ng DAMPs (damage-associated molecular paJerns). We further show that this func;onal state of the macrophages is partly directed by the metabolite lactate that is generated by the epithelial compartment of the skin in condi;ons of sterile inflamma;on. Remarkably, inhibi;on of the lactate-mediated metabolic crosstalk between the skin epithelia and macrophages leads to a substan;al reduc;on in the macrophage effector func;on and, in turn, inflamma;on. We further show that the principle of lactate metabolic crosstalk can be targeted to treat inflamma;on in the imiquimod-induced mice model of psoriasis. Finally, using a macrophage-depleted mouse model system we inves;gate the role of these cell types in regula;ng skin development. Our results suggest that macrophage-mediated remodeling of the skin ECM is essen;al for driving the development of skin appendages. These studies have elucidated the developmental and inflammatory roles of macrophages in the developing skin. Publica;ons and Patent: 1. Ayyangar U, Karkhanis A, Tay H, Afandi AFB, BhaJacharjee O, KS L, Lee SH, Chan J & Raghavan S (2024) Metabolic rewiring of macrophages by epidermal-derived lactate promotes sterile inflamma;on in the murine skin. EMBO J 2. BhaJacharjee O, Ayyangar U, Kurbet AS, Lakshmanan V, Palakode; D, Ginhoux F & Raghavan S (2021) Epithelial-Macrophage Crosstalk Ini;ates Sterile Inflamma;on in Embryonic Skin. Front Immunol 12. 3. BhaJacharjee O, Ayyangar U, Kurbet AS, Ashok D & Raghavan S (2019) Unraveling the ECM-immune cell crosstalk in skin diseases. Front Cell Dev Biol 7 doi:10.3389/fcell.2019.00068. 4. [Patent] Citric Acid Cycle and Lactate Transport Inhibitors for Preven;on and/or Treatment of Skin Disorders PCT Applica;on number: PCT/IB2022/061996 Date of Filing: 10.12.2022 Inventors: UJkarsh Ayyangar, Srikala Raghavan.

In this presentation I will discuss a new layer of regulation in development and repair – namely organellar heterogeneity. Many organelles perform pan-cellular functions and were hence considered to be rather similar between cell types. However, we have recently identified an unexpected heterogeneity of organelles, such as the centrosome, the nucleolus or the mitochondria between even closely related cell types, such as neural stem cells and neurons revealing or neurons and astrocytes. I will describe these discoveries and then talk about how this matters for development, disease and repair. I will highlight how the localization of a ubiquitous splicing factor at the centrosome matters for correct neuronal positioning and how localization can explain why mutation of a ubiquitous protein has an organ-specific phenotype. This will bring us to moonlighting proteins and I will share unpublished data on the role of a cytoskeleton-associated protein in the nucleus. In concluding I may briefly touch on mitochondrial heterogeneity and its role in direct neuronal reprogramming including human astroglia-to neuron conversion. Taken together, our knowledge about basic mechanisms of neurogenesis allowed making great strides towards neuronal repair.

Collagen II (COL2A1) is a crucial structural protein in cartilage extracellular matrix. COL2A1 mutations lead to various cartilage disorders, ranging from mild early-onset arthritis to lethal perinatal malformations. Existing in vitro disease models do not use disease-specific cells and mouse models do not replicate human physiology, making the study of collagen II disorders particularly challenging. Consequently, the molecular pathology of these mutations is not fully understood, and no effective drug therapies are available for patients. To tackle this problem, we have modelled a severe and a lethal cartilage disorder caused by heterozygous COL2A1 p.R989C and p.G1113C mutations, respectively. We introduced the patient mutations into hiPSCs using CRISPR/Cas9 gene editing. Mutant and isogenic control lines were differentiated into human cartilage organoids using our developed protocol, then we used these organoids to uncover molecular mechanisms of these mutations. While both mutations cause similar collagen biosynthetic defects, they are more severe in p.G1113C mutant organoids compared to p.R989C. The mutant organoids exhibited slow collagen II folding, intracellular retention, dilated endoplasmic reticulum (ER), and reduced and abnormal collagen II fibrils in the extracellular matrix, mirroring findings in patients and mouse models. Despite these similarities, the mutations affected chondrocyte maturation differently: p.R989C accelerated hypertrophy, whereas p.G1113C delayed it, potentially explaining the observed bone defects in patients. Using unbiased whole transcriptomics analysis, we found that both mutant collagen II proteins activate the PERK arm of the ER unfolded protein response (UPR), suggesting ER stress, consistent with the dilated ER found in the mutants. This study expands our understanding of COL2A1 molecular mechanisms in clinically relevant human disease models. Our in vitro models, which reproduce disease phenotypes, have significant potential for pre-clinical drug screening. Ultimately, we can therapeutically target the pathogenic pathway identified in this study, potentially leading to new therapeutic strategies for collagen II disorders.

Muscle tissue engineering holds significant promise for regenerative medicine, disease modeling, and cultured meat production. This field utilizes muscle cells, scaffolds to support these cells, and biochemical cues to guide their development. For muscle cells to mature into functional tissue, they must undergo myogenesis, which involves cell proliferation, followed by exit from cell cycle, differentiation, alignment, and fusion into elongated, multinucleated myofibers. An essential aspect of effective scaffold design is creating structures that promote proper cell alignment and fusion. In this study, we fabricated scaffolds from marine-derived biopolymers, including salmon gelatin, alginate, agarose, and glycerol, resulting in both flat and microstructured surfaces for comparative analysis. We assessed the impact of these microstructured scaffolds on muscle cell behavior, focusing on alignment, glycolytic metabolism, myogenic gene expression, and transcriptomic profiles. Muscle cells cultured on microchannel scaffolds developed parallel, elongated, multinucleated structures resembling muscle bundles, with elevated glycolytic metabolism, when compared to the use of flat scaffolds. Gene expression analysis revealed significant correlations between myogenic and fusion markers, such as Myomaker with MyoD, Myomixer with Myosin heavy chain, and Myogenin with Myosin heavy chain. Immunofluorescence confirmed the expression of these markers after seven days of culture. Transcriptome analysis using high throughput sequencing highlighted notable differences between flat and microstructured scaffolds. Functional enrichment analysis identified key gene modules related to muscle development, including filament sliding, muscle contraction, and sarcomere organization. This study enhances the understanding of scaffold design in muscle tissue engineering, providing insights into the underlying mechanisms that are benefit by muscle cell alignment. The findings suggest that microstructured scaffolds are essential tools for advancing muscle tissue engineering practices.

none

Macrophages are the earliest set of immune cells that populate the murine skin. However, much less is understood about their role in regulating developmental and inflammatory programs in the developing skin. Our study suggests that macrophages in the developing skin can respond to sterile inflammatory cues by augmenting the synthesis of ECM (extracellular matrix) remodeling enzymes called MMPs (matrix metalloproteinases). These MMPs exacerbate chronic inflammation by generating DAMPs (damage-associated molecular patterns). We further show that this functional state of the macrophages is partly directed by the metabolite lactate that is generated by the epithelial compartment of the skin in conditions of sterile inflammation. Remarkably, inhibition of the lactate-mediated metabolic crosstalk between the skin epithelia and macrophages leads to a substantial reduction in the macrophage effector function and, in turn, inflammation. We further show that the principle of lactate metabolic crosstalk can be targeted to treat inflammation in the imiquimod-induced mice model of psoriasis. Finally, using a macrophage-depleted mouse model system we investigate the role of these cell types in regulating skin development. Our results suggest that macrophage-mediated remodeling of the skin ECM is essential for driving the development of skin appendages. These studies have elucidated the developmental and inflammatory roles of macrophages in the developing skin. Publications and Patent: 1. Ayyangar U, Karkhanis A, Tay H, Afandi AFB, Bhattacharjee O, KS L, Lee SH, Chan J & Raghavan S (2024) Metabolic rewiring of macrophages by epidermal-derived lactate promotes sterile inflammation in the murine skin. EMBO J 2. Bhattacharjee O, Ayyangar U, Kurbet AS, Lakshmanan V, Palakodeti D, Ginhoux F & Raghavan S (2021) Epithelial-Macrophage Crosstalk Initiates Sterile Inflammation in Embryonic Skin. Front Immunol 12. 3. Bhattacharjee O, Ayyangar U, Kurbet AS, Ashok D & Raghavan S (2019) Unraveling the ECM-immune cell crosstalk in skin diseases. Front Cell Dev Biol 7 doi:10.3389/fcell.2019.00068. 4. [Patent] Citric Acid Cycle and Lactate Transport Inhibitors for Prevention and/or Treatment of Skin Disorders PCT Application number: PCT/IB2022/061996 Date of Filing: 10.12.2022 Inventors: Uttkarsh Ayyangar, Srikala Raghavan.

Cell shape changes play a fundamental role in development and disease, often accompanying transitions in cell state. Despite the importance of cellular shape for biological function, most studies investigating state transitions focus on gene expression changes. Here, we combine morphometric analysis with stochastic inference, mathematical modelling, and molecular perturbations to interrogate cell shape dynamics during epithelial-to-mesenchymal transition (EMT). We extract a low-dimensional representation of the morphogenetic landscape associated with EMT and show that EMT-associated cell spreading corresponds to a transition between cell shape attractors within this landscape. Strikingly, we observe a peak in cell shape noise at the time of spreading. Theoretical modelling and experimental perturbations show that shape noise plays a functional role in cell spreading, accelerating the transition between shape attractors. Together, our study characterizes the morphogenetic landscape of EMT-associated cell spreading, a framework that will be widely applicable to investigate shape changes in physiology and disease. Overall, our findings reveal that cellular noise is not merely a signature of cell shape changes but influences how they occur, offering new insights into the functional role of noise.

Acute liver failure (ALF) is a life-threatening condition with limited treatment options beyond liver transplantation in non-acetaminophen cases. The extensive loss of liver function results from severe hepatocyte death, where elevated reactive oxygen species (ROS) play a significant role. Nuclear factor erythroid-2 like 2 (Nrf2) is crucial in ROS defense by regulating genes such as glutathione peroxidase 4 (GPX4), which prevents lipid peroxidation (LPO). GPX4 is involved in several regulated cell processes, including apoptosis and ferroptosis. GPX4 expression was measured in liver samples from healthy individuals, ALF, and acute-on-chronic liver failure (ACLF) patients. To investigate GPX4's role, mice with hepatocyte-specific deletion of Gpx4 (Gpx4Δhepa) and both Gpx4 and the Nrf2 repressor, Keap1, (Gpx4ΔhepaKeap1Δhepa) were generated. ALF was induced in mice using a carbon tetrachloride (CCl4) and a bile duct ligation (BDL) cholestasis model, each lasting 48 hours. ALF patients exhibited reduced GPX4 levels compared to healthy individuals and ACLF patients, consistent with observations in CCl4-treated wild- type mice. ALF-induced Gpx4Δhepa mice exhibited increased hepatocyte death and liver dysfunction upon CCl4 treatment, with increased apoptosis despite no changes in LPO markers. Activation of Nrf2 in Gpx4ΔhepaKeap1Δhepamice reversed CCl4-induced damage, reducing necrosis and apoptosis markers while inducing anti-apoptotic BCL2. Our results demonstrate that Gpx4 plays a critical role in ALF as its absence leads to hepatocyte apoptosis. Activating Keap1-dependent pathways targeting antioxidant defense system and upregulating BCL2 provides substantial protection against ALF in mice lacking Gpx4 in hepatocytes. Our findings suggest that the Keap1-Nrf2 axis is a promising therapeutic target in ALF.

In this lecture we explore the evolution of the eukaryotic cell. As we will see, much of the machinery, including actin, tubulin and ESCRT-III, has its origins in archaea. Building on this, we will see how a better understanding of archaeal cell biology, and ESCRT-III dependent membrane remodelling machinery in particular, can shed light on the structure and function of its eukaryotic counterparts, and the evolution of the eukaryotic cell division cycle.

Adult primary human hepatocytes (PHHs) are the gold standard in ex vivo toxicological studies and possess the clinical potential to treat patients with liver disease as advanced therapy medicinal products (ATMPs). However, the utility of this valuable cell type has been limited by short-term functionality and limited expansion potential in vitro. While notable advances have been made in the long-term maintenance of primary hepatocytes, there has been limited success in driving the efficient generation and expansion of adult PHH-derived organoids which recapitulate both liver tissue architecture and function, hampering in vitro studies and regenerative medicine applications. Here we describe the mass generation and long-term expansion of hepatobiliary organoids with functionally interconnected hepatic and biliary-like structures from adult primary human hepatocytes. Hepatobiliary organoids retain the expression of lineage and functional markers, closely resembling PHH, while also acquiring the expression of regeneration, fetal and biliary markers. Organoids perform key hepatocyte functions while proliferating and can be matured to enhance their functionality.

Many natural structures such as proteins, climbing vines, and seashells exhibit a well defined chirality, some are left-handed, some are right-handed, some are both. The ultimate origin of chirality is one of Nature's great mystery. However, geometry and mechanics play a fundamental role in assigning chirality and carrying this information from microscopic to macroscopic scales. In this talk, I will discuss the general problem of chirality, chirality measure, and chirality transfer, trace its history, and use examples from chemistry and biology to obtain general principles with some surprising twists.

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. The members of the Transient Receptor Potential (TRP) channel superfamily of ion channels are characterized by large N- and C-terminal IDRs that mediate intra- and intermolecular contacts with other proteins and the lipid bilayer1. TRP channels play central roles in almost every (patho)physiological process, including thermo- and osmosensation, nociception and infectious diseases2. A molecular understanding of the polymodality of TRP channel regulation is thus crucial for a fundamental understanding of sensory perception and cellular responses to diverse stimuli. We hypothesize that the IDRs harbor regulatory elements that allow TRP channels to meaningfully integrate diverse signaling inputs. We recently generated the first full-length structural model of a TRP channel, TRP Vanilloid 4, that takes the dimensions of the IDR and its membrane-interactions into account3. Intriguingly, deletion of the channel’s N-terminal IDR, consisting of ~150 amino acids, renders TRPV4 unresponsive to osmotic stimuli, while distinct IDR regions enhance or subdue channel function. Together, these regulatory elements in the IDR modulate channel responses in a hierarchical, lipid-dependent manner. To enhance our understanding of TRP channel regulation by its large IDR, we also incorporated small molecules, proteins and disease mutations4 into our functional model of TRPV4. Furthermore, by exploring the structural dynamics and functional roles of the IDRs in other TRP channels, we find that multilayered signal integration by the IDRs is a widespread phenomenon in this important protein family. Accordingly, to understand TRP channel function, their often extensive IDRs cannot be ignored. Our work shows that “IDR cartography”, i.e., mapping structural and functional properties onto distinct IDR regions through an integrated structural biology approach, sheds light on the complex regulation of membrane receptors through their hitherto mostly neglected regions and provides a framework for signal integration on the molecular level.

Implantation of the early embryo into the uterine wall is a key step in the mammalian reproductive cycle. This critical process mediates the connection between the embryo and the mother during the early stages of pregnancy. As the uterine tissues conceal the implanting embryo, the cellular interactions at the embryo-maternal interface are inaccessible for direct analysis and therefore poorly understood. Moreover, some mammalian species can 'pause' the development of their embryos just before implantation. These embryos enter a reversible state of dormancy (diapause) for extended periods, delaying the time of birth. How the dormant embryos remain in suspended animation without compromising their developmental potential is still obscure. Using the mouse as a model system, our lab aims to decipher the cellular mechanisms of embryonic development and dormancy and the dynamic crosstalk between the implanting embryo and the mother.

All extant cells known to humankind build proteins from the same 20 coded amino acids. Is this canonical alphabet a prerequisite for life to be as successful as it has been on our planet or could protein structures and functions depend on a different/smaller set of amino acids? We report structural and functional propensities of proteins and peptide libraries built from different subsets of both canonical and non-canonical amino acids, mimicking different hypothetical stages of the prebiotic-to-biological sequences. The study of origins of life implies that earlier cells functioned with a smaller alphabet, which was selected from a pool of prebiotically plausible (canonical and non-canonical) amino acids before the fixation of the Central Dogma. Our work implies that the “early” canonical amino acids that were selected from the prebiotic environment have a higher structure-forming propensity than possible alternatives. Despite lacking positively charged and aromatic residues, proteins composed from such “early” components would be prone to structure formation but also capable of interacting with organic and inorganic cofactors. On select examples, we observe that such binding can significantly assist with early protein folding as well as with catalytic and binding propensities. Our work indicates that protein folding propensity was an important factor during the earliest stages of the genetic code evolution. A reduced acidic alphabet would be sufficient to build proteins, capitalizing on interactions that are less frequent or rare in today’s biology.

During embryonic development, unspecialized cell masses are transformed into complex tissues and organs through coordinated movements and interactions, driven by morphogenesis and cell fate acquisition. This study aims to elucidate how these processes are coordinated in space and time using the Xenopus mucociliary epithelium as a model. We will first provide an overview of the dynamics of cell fate acquisition, followed by an examination of the morphogenetic processes that accompany these fate decisions. Finally, we aim to develop a predictive model of cell fate acquisition by linking cellular behavior with cell fate decisions. This research will advance our understanding of the intricate relationship between cell fate and tissue morphogenesis, offering insights into the fundamental principles of tissue formation.

The perpetuation of bacteria in multi-species communities is affected by how species interact. In particular, bacterial populations may employ mechanisms that enable multiple species to coexist. We investigate the interactions between two opportunistic pathogens, P. aeruginosa and S. aureus, on a porous surface. Using an imaging method that we recently developed, Imaging using Reflected Illuminated Structures (IRIS), we find that both species coexist and proliferate but remain physically segregated due the interactions between surfactant produced by P. aeruginosa and amyloid fibrils produced by S. aureus. This interaction gives rise to dense single-species populations that are spatially separated. Our findings suggest that surfactant interactions can have a critical role in determining how bacterial species interact and whether they can coexist. To complement our understanding of bacterial colonization, we have investigated mechanisms that mammalian hosts employ to eradicate bacteria. We have identified that histones, which organize DNA in mammalian cells, synergize with pore-forming antimicrobial peptides to kill bacteria. We find that the entry of histones into bacteria re-organizes their chromosomes and inhibits transcription, leading to bacterial death. Our findings suggest a novel antimicrobial mechanism that is achieved through differential targeting by histones and antimicrobial peptides. This synergistic mechanism can potentially be exploited to establish a new class of potent antimicrobials.

Synthetic biology and metabolic engineering applications in microalgae: Sustainable Biotechnology at KAUST

Mammalian development is still enigmatic processes as it undergoes all the process in mother's uterus. Historically, simply taking any part of the processes out from the uterus to dish has brought tremendous findings. For example, in vitro fertilization and the subsequent culture of embryos up to the blastocyst stage have given us the opportunity to monitor, manipulate, and investigate post-fertilization development.Here we will introduce two in vitro model systems: directly induced oocyte-like cells (DIOLs) and human advanced gastruloid model, which recapitulates mouse oocyte development and human post-gastrulation development, respectively, entirely in vitro. These systems provide robust and scalable platforms for investigating the mechanisms underlying mammalian life cycles, as well as failures in these processes, including infertility and developmental diseases.

One of the grand challenges in synthetic biology is the construction of selectively open molecular systems integrated into cell-like containers with control of solute fluxes and a constant supply of energy to fuel ATP-requiring processes. Such systems should enable long-term metabolic energy conservation and physicochemical homeostasis and a better understanding of how living cells perform these tasks. In living cells metabolic reaction networks orchestrate the supply, regeneration, and removal of energy. These energy modules are responsible for the almost flawless exertion of all cellular functions, and thus, sustaining life. In contrast, the prolonged performance of man-made non-equilibrium systems is often limited by the availability of fuel molecules or the accumulation of waste products due to a lack of control mechanisms. Here, we present synthetic vesicle systems that allow for the compartmentalization of ATP-driven non-equilibrium processes and the recycling of ATP by means of the continuous import of ATP into the lumen and concomitant export of ADP from the lumen. The ATP and proton motive force generating networks we developed for studying metabolic processes in vesicles perform at least an order of magnitude better than other systems described so far. They enable control of the volume, osmotic pressure, ionic strength and pH of the vesicles, and allow for cross-feeding of ATP between different populations of vesicles, and the synthesis of lipid precursors and membrane growth.

Lipids are metabolites with enormous structural and functional diversity. In biological systems, they function as sources of energy, form physical barriers, and orchestrate cellular signaling and trafficking in manifold ways. Because of the large number of lipids, complex metabolic networks, small size, and physicochemical properties, the elucidation of their biological functions has been challenging. I will present on chemical biology approaches that address some of these challenges by expanding our ability to control lipid function with high spatiotemporal precision, to target membrane proteins including Ras, Rho, and Rab GTPases, and to modulate protein-membrane interfaces with novel pharmacology.

The soil dwelling bacterium Myxococcus xanthus is an amazing organism that uses collective motility to hunt in giant packs when near prey and to form beautiful and protective macroscopic structures comprising millions of cells when food is scarce. I will present an overview of how these cells move and how they regulate that motion to produce different phases of collective behavior. Inspired by recent work on active matter and the physics liquid crystals, I will discuss experiments that reveal how these cells generate nematic order, how defect structure can dictate global behavior, how transient polar states govern the ultimate dynamics, and how cells actively tune the Péclet number of the population to drive a phase transition from a gas-like flocking state to an aggregated liquid-droplet state during starvation.

Artificial intelligence (AI) techniques led to impressive advancements in many disciplines and are transforming science and societies. In structural biology the accurate predictions of protein structure from amino-acid sequences are clearly disruptive and have become invaluable tools for structural biology research. However, despite their undisputable usefulness they do have fundamental limitations in providing insights into, for example, the determinants of protein complex formation, protein conformational switching and resulting functional dynamics, let alone an adequate understanding of intrinsically disordered proteins (IDPs), amongst other aspects of protein biology. However, these AI-inherent limitations underscore the significance and future relevance of nuclear magnetic resonance (NMR) as a crucial experimental tool in filling these crucial knowledge gaps. IDPs are challenging the established structural biology structure-function paradigm and thus mandate suitable theoretical framework and concepts to properly address the subtle interdependence between protein structure and dynamics. In the lecture it will be described how the combination of NMR and novel computational protein sequence analysis tools can be used to provide a more comprehensive view of IDPs and how they can simultaneously maintain modularity, robustness, and adaptability, as essential features for their biological functionalities. Additionally, NMR avoids the fundamental shortcomings of conventional Structure-Based-Drug-Design (static snapshot, lack of dynamics, no direct and quantitative information about intermolecular interactions) by providing access to site-specific information about structural dynamics and the chemical environment in solution. In the lecture it will be outlined how previous technical limitations can be overcome thanks to the advent of sophisticated isotope-labeling strategies and by applying meaningful NMR experiments. The combination of these techniques with AI-based approaches for protein structure predictions enables the consideration of the entire conformational state distribution of protein-ligand complexes and to effectively harness enthalpy-entropy compensatory effects, with considerable impact on future drug design strategies.

Fibroblasts are stromal cells of connective tissue, which are critical for organ development, homeostasis and disease. Single cell transcriptomic analyses have revealed a great level of inter- and intra-organ heterogeneity of fibroblasts, however, the functional implications and lineage relations of different fibroblast subtypes are unknown. In the mammary gland, fibroblasts support all stages of development, including branching morphogenesis, as well as the transition into breast cancer. Here we characterized pubertal mammary fibroblasts using single cell RNA sequencing, spatial mapping and in vivo lineage tracing and discovered a transient niche-forming population of contractile fibroblasts that specifically localize around growing epithelium after being recruited from the surrounding fat pad. Using functional organoid experiments we revealed that fibroblast contractility is needed for epithelial morphogenesis and that different fibroblast populations are activated by contact with the epithelium to support its morphogenesis. In summary, our detailed characterization brings new insight into mammary fibroblast heterogeneity, including functional relevance for epithelial morphogenesis and lineage hierarchy during normal mammary gland development in mice.

Mitochondrial architecture is characterized by an extended and elaborately folded inner mitochondrial membrane (IMM). Due to their spatial constraints and specific protein inventory, the cristae domains of the IMM shape a unique micro-compartment optimized for oxidative phosphorylation. Crista junctions (CJs) form the highly curved neck regions of cristae and have been suggested to act as selective entry gates into the cristae space. The mitochondrial contact site and cristae organizing system (MICOS) constitutes the backbone scaffold of CJs, but molecular insights have remained sparse due to a lack of structural information. Here, I provide an overview on our efforts to characterize structure and function of crista junctions using an integrative structural biology approach.

Archaea constitute one of the primary domains of life. Although many archaea are found in extreme environments, they are ubiquitous in almost all habitats. They play significant roles in the carbon and nitrogen cycling of the earth and are the only producers of the climate gas methane. It is, therefore, surprising how scarce the knowledge about their cell biology is. This is mainly due to the difficulties in establishing archaeal model systems where genetics can be done efficiently. The present advances in our knowledge about archaeal cell division systems (ESCRT III- and FTSZ-based) and the role and structure of archaeal cell surface appendages will be discussed

The organization of the cell is often hypothesized rather than directly observed. With the rise of super-resolution methods, reaching a novel understanding of cell organization is now possible. In this seminar, I will first show how STED let us redefine the organization of the adherens junction and polarity proteins in the human intestinal epithelium. Then, I will discuss our ongoing effort using DNA-PAINT to uncover the organization of the developing muscle at 5 nm resolution.

Small RNA-mediated silencing pathways are essential players in the relentless genetic conflict between transposable elements and host genomes. In animal gonads, the PIWI-interacting RNA (piRNA) pathway enforces effective transposon silencing in the cytoplasm and nucleus. Conceptually similar to prokaryotic CRISPR/Cas systems, the piRNA pathway uses effector proteins complexed with single-stranded guide RNAs (22-32nt piRNAs) derived from genomic loci, which act as adaptive and heritable sequence repositories. In my seminar, I will focus on the adult ovary ‘ecosystem’ in Drosophila melanogaster and provide an overview of our current understanding of the piRNA pathway. I will discuss how the rare event of an LTR element gaining infectivity allowed a genuine endogenous retrovirus to temporarily escape piRNA-mediated silencing, resulting in a profound example of retroviral diversification, and how the host responded.

Mammalian development is a highly orchestrated process where millions of cells move in concert to ensure the formation of diverse tissues and organs. In particular, during gastrulation and early organogenesis, a relatively homogenous group of pluripotent cells rapidly diversify to shape complex morphological domains with specialized functions. These intricate processes require multiple regulative layers that dynamically crosstalk to guarantee developmental robustness, including the interactions between genes, cells, and the extracellular environment. Despite foundational work in the past century, quantitative measurements of these dynamic interactions remain challenging, mainly because of the embryo inaccessibility in the maternal womb and the lack of adequate tools to decode its complexity. I will present novel stem cell-based and genomic approaches that we developed to make mammalian embryogenesis more accessible and tunable, including enhanced in vitro models, 3D spatial transcriptomic maps, and single-cell genetic lineage tracing technologies. This innovative toolkit allowed us to assess the role of the extracellular matrix in tissue morphogenesis, study the spatial organization of gene expression in mutant embryos, and chart complex developmental trajectories during critical windows of mammalian development.

Flows in the fluidic interior of living cells can serve biological function or act as signatures of how intracellular forces are exerted. I'll discuss examples of each. One is understanding the emergence of cell-spanning vortical flows in large developing egg cells, while the other arises in studying the nature of force transduction in single cell embryos moving towards their first cell division. Both involve the cytoskeleton, that set of polymers, cross-linkers, and molecular motors that underlie much of the active mechanics within cells, and has led to the development of new coarse-grained active matter models and novel instabilities.

Living tissues are characterized by an intrinsically mechano-chemical interplay of active physical forces and complex biochemical signalling pathways. Either type of feature alone can give rise to complex emergent phenomena at the tissue scale, for example mechanically driven flocking or rigidity transitions, or chemically driven reaction-diffusion instabilities. An important question is thus how to quantitatively assess the contribution of these different cues to the large-scale dynamics of biological materials in cases where multiple phenomena are present or even coupled. In this talk, I will present a few examples that we have worked on in the context of collective cell migration and organoid morphogenesis.

The spontaneous generation of patterns and structures occurs in many living systems and is linked to biological form and function. Such processes often take place on domains which themselves evolve in time, and they can be guided by or coupled to geometrical features. Using two different biophysical examples, I will discuss how geometry directs spatial organization in cellular and multicellular systems. I will discuss how boundary geometry controls bulk organisation in the developing mouse epiblast, and how contact area-dependent signaling interplays with cell shape dynamics.

SWIR provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. In this wavelength range tissues become translucent. Recent progress in detection technology and the development of probes demonstrated that, in principal, SWIR imaging enables applications which were previously not feasible with any other technique. These advantages will enable new capabilities in preclinical imaging. While the optical advantages are clear, the successful translation of SWIR imaging into routine applications requires novel, bright and targeted probes as well as advanced imaging setups. Most SWIR imaging setups so far are used for proof of principal demonstrations only. To utilize the full potential, our first goal is developing novel SWIR imaging setups, which enable high-speed intravital imaging, ultra-sensitive whole animal imaging and fluorescence molecular tomography in mice in the SWIR. Our second goal is to develop novel bright and targeted SWIR probes for preclinical research in oncology. The novel applications include SWIR imaging of physiology and metabolic activity and targeted SWIR imaging of tumors. SWIR intravital microscopy will allow imaging the brain vasculature around tumors in mice through intact skin and skull and generating detailed blood flow-maps in mice. In the future, advantages of SWIR imaging will also improve fluorescence guided surgery and other clinical applications in precision medicine. To advance clinical research and enable clinicians to utilize and benefit from the great potential of SWIR imaging, the overarching goal of us is to develop novel non- toxic SWIR probes for future clinical use. The unprecedented sensitivity of SWIR imaging in combination with its deep penetration and high resolution should allow detecting cancer cells with very high sensitivity, which is an ultimate goal for a surgeon.

The neural crest is a multipotent population in the vertebrate embryo, which arises from the neural border, undergoes EMT at the end of neurulation, and migrates to different parts of the embryo. At their final destinations, the neural crest cells differentiate into a host of different derivatives, such as neurons, glia, melanocytes and connective tissue, making the neural crest an important contributor of embryogenesis. Although many genes controlling these different processes have been individually studied, the complete gene-regulatory-network remains to be elucidated. In our lab, through the intersection of different high-throughput techniques like scRNAseq and ChIPseq, not only have we identified potential regulators, but also validated some of their functions on a large scale. In the poorly-studied premigratory neural crest population, we have observed early fate predispositions in the neural crest population, as well as a retention of multipotency characteristics till late neurula stages. Further, at earlier (gastrula) stages, we have also identified novel candidates driving the fate specification of the neural border into the neural crest and the cranial placodes. On the other hand, we also use candidate-based approaches to study the mechanisms of function of the identified genes in greater detail, such as Prdm12, a transcription factor with H3K4 methyl transferase activity. During early neurula stages, Prdm12 restricts the expression of neural crest genes to establish the boundaries of the different ectodermal domains. However, at a later stage, we observe that Prdm12 affects neural crest migration at a later stage through modulating the expression of cadherins and the Wnt pathway. Through my talk, I will present an overview of how we use these multiple different approaches to study the different genes at the scale of a regulatory network.

Sexual reproduction is an ancient and conserved feature of life on earth, but the mechanisms that determine the sex of an individual are mesmerizingly diverse and have had rapid turnover rates during evolution. Do sex determination genetic pathways converge in different organisms? What drives sex chromosome evolution? What are the mechanisms underlying switches between sex determination systems? Are all sex chromosomes equal in terms of origin and evolutionary trajectories? The answers are complex but the ongoing genomic revolution and the use of ‘alternative’ model organisms is shedding new light on sex determination molecular mechanisms, sex chromosome diversity and evolution. I will describe how brown algae, which have been evolving independently from animal and plants for more than a billion years, are contributing to this dynamic field of research.

Necroptosis is a tightly regulated form of cell death that plays a critical role in the pathogenesis of various diseases, including neurologic, cardiovascular, as well as numerous inflammatory diseases. During necroptosis, affected cells exhibit membrane rupture, resulting in the release of intracellular components. The activation and execution of necroptosis involve a complex series of biochemical reactions within the cell. The classical necroptosis signaling pathway is primarily initiated by stimuli from the tumor necrosis factor family, such as TNF-α, which induce necrotic cell death through the RIPK1-RIPK3-MLKL pathway. Recently, our study has uncovered an additional mechanism of necroptosis activation in response to extracellular osmotic stresses. Notably, unlike previously identified necroptosis inducers, osmotic stress triggers necroptosis by directly activating RIPK3 kinase activity, which is facilitated by an increase in cytosolic pH mediated by the Na+/H+ exchanger SLC9A1. SARM1 is a well-known mediator of axonal degeneration and has been reported to deplete NAD+ levels following nerve injury through its NADase activity. Studies have shown that deficiency of SARM1 can attenuate axon degeneration in various experimental models. Here, we have identified a class of potent SARM1 activators. These activators have been observed to induce SARM1-dependent cell death and axon degeneration in 293T cells and dorsal root ganglia (DRG), respectively, at specific concentrations. Interestingly, unlike previously reported SARM1 activators that interact directly with the ARM domain, we found that disruption of the NMN/NAD+ binding site within the ARM domain does not hinder the ability of this activator to induce SARM1 activation. Instead, this activator activities SARM1 NADase through a liquid-to-solid phase transition. This finding suggests a model in which SARM1 condensation leads to NADase activation and subsequent axon degeneration. Notably, this mechanism shares similarities with the activation behavior observed in Bacterial TIR enzymes during their antiphage responses.

Oxygen is the fundamental electron acceptor in metabolism. As a byproduct of aerobic respiration, various reactive oxygen species (ROS) are formed. The balance of oxygenation and ROS is a critical aspect of homeostasis. We present the concept of using electrochemical methods to manipulate the levels of oxygen and ROS in biological systems in order to produce a desired outcome. Extensive oxygen depletion combined with generation of large concentrations of ROS can lead to cell death, which we propose is a novel and useful approach to precise electrosurgery of pathogenic tissues. Lower levels of oxygen reduction/ROS generation can alter the function of numerous biological pathways. Of particular interest is targeting ion channels using locally-generated hydrogen peroxide. This offers a completely new method to alter electrophysiology in vitro or in vivo. I will present several examples of faradaic electrochemistry on oxygen acting at the level of different targets. Electrochemical approaches can be used to precisely deoxygenate a given environment, or combine deoxygenation with generation of ROS. A further level of electrochemical control is using electrolysis to interrupt electrophysiological activity via rapid local changes in pH. Examples of nerve lesioning in animal models will be shown.

Synthetic chemistry has enabled the creation of materials with remarkable properties, yet these materials often lack the dynamic nature exhibited by biological systems. In contrast, living matter is self-organizing and responsive, which is critical for many processes, including cell differentiation, sensing, transport, actuation, structural support, and—more generally—adaptation to internal and external stimuli. Intriguingly, the utilization of DNA nanotechnology principles in synthetic materials has opened avenues for achieving a spectrum of features and a degree of control reminiscent of biological systems. These materials have begun to emulate key cellular mechanisms, including the modulation of viscoelastic properties in the extracellular matrix, shape changes of the cytoskeleton, control over molecular transport, and localization of processes in biomolecular condensates. In this talk, I will describe our progress in developing such programmable materials and underscore their significance in two specific application domains. Firstly, I will highlight a new precision matrix for the culture of cells and organoids. By integrating customizable mechanics and predictable responsive features, these matrices help guide and interrogate cellular development and morphogenesis. Secondly, I will describe our work on intelligent polymers designed for streamlining nucleic acid sequencing and pathogen detection.

Lung cancer is the leading cause for cancer-related deaths worldwide with limited treatment options. Thus, new orthogonal treatment options are urgently needed. The cytochrome P450 4F11 (CYP4F11) is heavily upregulated in lung cancer patients. CYP4F11 catalyzes the w-hydroxylation of arachidonic acid yielding 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent lipid mediator and regulates blood pressure and angiogenesis in healthy individuals. In cancer, 20-HETE signaling leads to cell proliferation and migration and tumor angiogenesis. The Brixius lab has recently shown that a transient knockdown of CYP4F11 in lung cancer cell lines dramatically attenuates cell proliferation and migration, thus, demonstrating the high potential of CYP4F11 as drug target. We use a combination of cell biology, biochemistry, and X-ray protein crystallography to reveal structure and function of CYP4F11 and accelerate its use as lung cancer drug target for transformative therapeutics.

Gametic compatibility between different species is determined by the species specificity of their sperm and egg surface proteins, ultimately enabling or preventing hybridization. The egg membrane protein Bouncer is both necessary and sufficient for species-specific fertilization in medaka and zebrafish: changing only Bouncer on one species’ egg allows fertilization by the sperm of the other. Leveraging this specificity, we uncovered distinct amino acid residues and N-glycosylation patterns that differentially influence the function of medaka and zebrafish Bouncer and contribute to cross-species incompatibility. Equipped with novel Bouncer-mediated hybrids between medaka and zebrafish, we further explored how the timing of zygotic genome activation (ZGA) is regulated, given the 5-hour difference in ZGA onset in these species. Combining transcriptomics, chromatin accessibility profiling, and proteomics, we demonstrated maternal cytoplasmic dominance of ZGA timing and identified a potential novel regulator of vertebrate ZGA in medaka that may contribute to species-specific ZGA onset

A cell’s ability to sense and respond to physical cues is a specialized trait necessary to maintain tissue and organ homeostasis. In contrast, disruptions in local mechanical forces are linked with major chronic human health burdens, such as altered shear stress triggering cardiovascular disease and high velocity and magnitude compression resulting in traumatic brain injury. Decades of research have given insight into the specific consequences of physical cues on specialized cell functions. However, there remains an unmet need to understand how biophysical cues couple to molecular effects at a system-scale. This talk will present a novel computational tool to screen for molecules involved in cell signaling in response to specific biophysical cues. This approach enabled molecule identification, putative mechanosignaling network formation and cross-referencing to known cardiovascular disease indicators. We contrast this computational approach with an experimental one, in which we employed a microfluidic-based MEMS device to apply rapid, abrupt compression to individual neural stem cells and assess the cellular response to traumatic injury. Transcriptome measurements of cells compressed at varied magnitude and duration revealed a novel molecular signature of traumatic injury and a role for compression-induced signaling to alter neuroinflammation. Taken together, these data highlight the potential of systems biology approaches to contribute to study of mechanosignaling and elucidate the role of biophysics in regulating normal cell development and function.

What do journal editors do all day? Why is peer review so complicated? Do I need to worry about formatting my manuscript? How do you get into an editorial career? Find out the answers to these questions, and more, in this talk where I will give an overview of my day-to-day work (it's mostly reading), how scientific publishing works from an editorial perspective, and my career path from PhD to now.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as preeclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of its epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signaling controls human placental expansion and differentiation. NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator Mastermind-like 1 (MAML1). Doxycyclin-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, the differentiated hormone-producing cell type of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signaling plays a key role in human placental development promoting self-renewal of CTB progenitors.

The dynamics of biological networks enables the function of a variety of systems, including metabolic, gene-regulatory and neural circuits. To date, it remains unclear how to extract key features of networks if only time series data from (some) units are available. Here we report on recent progress on detecting structural features from observed dynamics. First, we demonstrate how to identify the number N of dynamical variables making up a network -- arguably its most fundamental property -- from recorded time series of only a small subset of n<N variables. Second, we sketch approaches to uncover network topological features from observed nodal time series data. We demonstrate first steps of applying the general theoretical methods developed to simulated models of biological and artifical systems. This is work with Jose Casadiego, Mor Nitzan, Hauke Haehne, Georg Boerner and others. [1] Topical Review: Marc Timme & Jose Casadiego, J. Phys. A 47:343001 (2014). [2] Casadiego et al., Nature Comm. 8:2192 (2017). [3] Nitzan et al., Science Adv. 3:e1600396 (2017). [4] Haehne et al., Phys. Rev. Lett. 122:158301 (2019).

The purely natural disaster, for which no one is to blame, is an Enlightenment category. Previously, there just disasters, and plenty of blame to go around: human, divine, and natural causes were all part of the explanation. Enlightenment thinkers (especially Enlightenment theologians) succeeded in separating causes of disasters into three mutually exclusive categories: natural causes (tragic, but no one’s fault); supernatural miracles (a divine suspension of the natural order); and human agency (the only category which was blameworthy). In the age of anthropogenic climate change, we are fast losing the category of the natural disaster – and with it, the concept of blameless evil.

Elucidating the developmental processes of organisms requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by trajectory reconstructions. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enabling in-silico fate mapping experiments. Finally, to demonstrate the versatility of our multi-modal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of Neuro-Mesodermal Progenitors (NMPs). Our publicly accessible web-based platform, Zebrahub, is a foundational resource for studying developmental processes at both transcriptional and spatiotemporal levels, providing researchers with an integrated approach to exploring and analyzing the complexities of cellular lineages during zebrafish embryogenesis. This work was made possible by the development of a broad range of technologies developed by my team, such as the napari viewer, novel state-of-the-art fluorescence microscopes (DaXi), self-supervised learning for bioimaging (CytoSelf), fast and scalable image processing for light-sheet time-lapse data (DEXP), high-accuracy cell tracking at scale in developing embryos (ultrack), fast, robust, and user-friendly image denoising (Aydin), and more recently the use of large language models for accelerating the dissemination of bioimaging skills and methods (Omega).

Until system-wide, unbiased analyses increased the number of recognized RNA-binding proteins (RBPs) well into the four-digit range, fewer than half of all RBPs were known (Castello et al., 2012; Hentze et al., 2018). This surprising outcome delivered new challenges for exploration: from the prevalence of structurally disordered RNA-binding regions with roles in the formation of RNA-protein condensates (Castello et al., 2016) to unsuspected and potentially pervasive connections between intermediary metabolism and RNA regulation (Beckmann et al., 2015). Riboregulation, the direct control of protein function by RNA, has begun to emerge as a new paradigm of biological control (Horos et al., 2019; Huppertz et al., 2022). We are beginning to understand molecular mechanisms of riboregulation, and investigation of the RBPs of mouse organs points to their broad roles in mammalian physiology (Perez-Perri et al., 2023). I will discuss these new facets of the RBP World and their implications for cell biology, metabolism and disease mechanisms.

Mitotic centrosomes are formed when centrioles recruit pericentriolar material (PCM) around themselves. The PCM comprises several hundred proteins, and its physical nature is hotly debated. In flies, Spd-2/CEP192 recruits Polo/PLK-1 and Cnn/CDK5RAP2 (fly/human nomenclature) to the centriole, and these form a solid-like scaffold that recruits PCM clients. Here we show that Spd-2 also recruits a liquid-like scaffold to centrioles that depends on Aurora A and TACC and is analogous to the liquid-like spindle domain (LISD) in mammalian acentrosomal meiotic spindles. The Polo/Cnn and Aurora A/TACC scaffolds can assemble and recruit PCM clients independently of one another, but centrosome function is severely perturbed in the absence of either scaffold. Thus, mitotic centrosomes in flies assemble upon co-existing solid- and liquid-like scaffolds organised by Spd-2/CEP192 and either Polo/PLK1 or Aurora A, respectively.

To improve the predictive power of in vitro models, vascular biology needs to be integrated into a functional, preferably facile, technology. The use of human induced pluripotent stem cells (hiPSC) in combination with micro-physiological systems, have emerged as tools to provide insight into the mechanisms of metabolic liver disorders, and liver fibrosis. A novel on-chip in vitro differentiation and tissue engineering procedure was used to generate a stem cell-based derived liver-on-chip model using a hiPSC panel of healthy and NASH donors in a 3D format. Liver fibrosis disease modeling was based on the stimulation of the model with transforming growth factor &#946;. The fibrosis phenotype was validated based on endothelial cell dysfunction, stellate cell activation, and deposition of extracellular matrix. Isogenic LoC models would facilitate the study of patho-physiological processes involved in liver disease progression for personalized medicine approaches.

During embryonic development, tissues evolve into intricate collectives that combine different specialized cell types. To create such complex ensembles, specialized precursor cells often move to integrate the target tissues where they execute their function. While much is known about the biochemical signals controlling cell movement in vivo, comparatively little is known about how mechanical stimuli from the environment direct migrating precursors. To address this question, we focus on the specialized precursor cells integrating the epidermis of the Xenopus embryo, using quantitative live imaging and mathematical modeling to characterize this process. Here, we describe how, during integration, the multiciliated cell (MCC) precursors extend actin-based filopodia directed at the epithelial vertices of neighboring epidermal cells. As the integrating precursors interact with their neighbors, they pull on the epithelial vertices through a mechanism that depends on the force generating motor myosin-II and the activity of a novel regulator of cell integration, LSR. To interpret our in vivo findings, we have designed a theoretical framework that models the physical environment of the epidermis during precursor integration. Our model and experimental data show that MCC precursors pull at the epithelial vertices to probe the local mechanical properties and identify suitable positions for cell integration. This pulling mechanism also equips MCC precursors with the ability to actively remodel their neighbors, and effectively generate a permissive environment that facilitates integration. Altogether, our work defines a novel durotaxis-like mechanism driving the integration of specialized precursors within a developing tissue, and highlights how individual migrating precursors can act as drivers of morphogenesis.

Cells in multicellular organisms are generally organized along the organisms’ axes to ensure coordinated cell division orientation, shape and differentiation. Central to this process is the establishment of cellular polarity, through local cortical accumulation of polarity proteins. While in animals and fungi, cell polarity mechanisms are relatively well-understood, it is largely unknown how cells are polarized in the plant kingdom. By studying the early plant embryo, our team identified a family of novel SOSEKI polarity proteins, deeply conserved among plants, and sharing functional domains with animal Wnt/polarity proteins. Characterization of these proteins revealed that regulated protein oligomerization and polymerization is a key principle that organizes cell polarity across kingdoms. In this seminar, I will focus on what we can learn about general organizing principles in cell polarity from comparative, structural, proteomic and functional analysis of this novel family of polarity proteins. I will address mechanisms underlying regulated polymerization, polar membrane targeting, and the control of polarity and development by mechanical signals.

Successful control of biological systems hinges on understanding their responses to both external stimuli and mutations. Our work along these lines focuses on the highly conserved Extracellular signal Regulated Kinase (ERK) cascade. Our research strategy relies on increasingly precise perturbations of this cascade in the fruit fly Drosophila melanogaster, which was instrumental in delineating key signaling systems. The first set of results is related to the large-scale effects of acute optogenetic pulses of ERK activation. Our phosphoproteomic analysis of the effects of these pulses revealed that ERK signaling controls synchrony of the cleavage cell cycles. The second set of results comes from work with flies that have been gene-edited to harbor deleterious mutations identified in humans. Our statistical analysis of defects in the ERK-dependent anatomical features in these flies suggests that part of the phenotypic heterogeneity in developmental abnormalities associated with deregulated ERK signaling is of stochastic origin. The presented approaches to studying signaling systems’ responses to static and dynamic perturbations promise to improve our chances of their control in a wide range of biomedical applications.

To generate the complex expression patterns required to build multicellular organisms, genes decode multiple signals to precisely tune their transcriptional output. A gene's cis-regulatory landscape, usually consisting of a series of regulatory elements (RE), must thus sense the activity of multiple transcription factors (TF) and integrate them in a specific manner. To investigate information processing, we use the Xist gene as a model, which governs X-chromosome inactivation. Xist integrates information on X-chromosomal dosage and developmental stage to trigger X inactivation during early embryonic development in females only. By combining various CRISPR screening approaches we dissect how Xist’s cis-regulatory landscape integrates input from a series of transcription factors to establish the correct expression pattern.

During vertebrate embryonic development left-right symmetrybreaking is innated by a ciliated organ called the Node. Within the Node, a leftward flow of extraembryonic fluid named the Nodal flow mediates the asymmetric expressions of Nodal factors. Although downstream Nodal pathway components leading to the establishment of the embryonic leftright axis are well known, less is known about the development and formation of the embryonic Node itself. Here, we reveal a novel role for the Meteorin protein family in the establishment of the left-right axis and in the formation of the Kupffer’s vesicle, the Node equivalent structure in zebrafish. The CRISPR/Cas9 genetic inactivation of each or all three zebrafish Meteorin members family (metrn, metrnl1 and metrnl2) led to defects in the properties of the Kupffer’s vesicle (KV) caused by an impaired assembly and migration of the dorsal forerunner cells (DFCs) that shape the KV. As a consequence, we show that Metrns loss-of-function results in disturbed Nodal factors expression, notably leading to heart looping defects. We demonstrate that through the genetic interaction with the Integrins Itg&#61537;V and Itg&#61538;1b, Metrn proteins regulate the DFC clustering. Together, our results identify a new role for the Meteorin protein family in the left-right asymmetry patterning during early embryonic vertebrate development. With the goal of identifying Metrns putative receptor(s), we are currently investigating the cellular pathway(s) in which Meteorin proteins are involved in and are developing approaches to validate the identified candidates as bona fide Metrns receptor(s).

Embryogenesis is brought about by the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key functions in germ layer specification and morphogenesis. Yet, how these pathways function in concert to render embryo patterning and morphogenesis so robust that key developmental processes unfold relatively normally even outside of the full embryonic context, is still not sufficiently understood. By analyzing ex vivo axis elongation in zebrafish embryonic explants, we show that Nodal signaling not only triggers axis elongation by induction of mesendodermal progenitors but also suppresses BMP signaling activity at the site of mesendoderm induction and thus explant elongation. Ectopic BMP signaling in the mesendoderm blocks cell alignment along the main explant axis and oriented mesendoderm intercalations, key processes to drive explant elongation. Translating these ex vivo observations to the intact zebrafish embryo showed that, similar to explants, Nodal signaling renders the dorsal domain less sensitive towards BMP signaling to allow effective cell intercalations, thereby conferring robustness to embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis extension by both inducing mesendoderm and maintaining low levels of BMP signaling activity in the dorsal portion of the mesendoderm.

Engineering Microenvironments for Organoid Architecture: The Power of Simple Tools in Recapitulating System Complexity In vitro 3D and organoid culture methods that mimic the rich complexity of cell population and extracellular matrix (ECM) components of in vivo tissues contribute greatly to furthering our understanding of various biological phenomena. Nevertheless, it remains a challenge to exert control over complex shape and architecture, as well as tissue-tissue interactions of cultured organoids. The current organoid development is highly depending on cellular self-organization, but the homogeneous culture conditions in vitro cannot provide spatial information to cells correctly. Engineering principles, on the other hand, are great tools that enable us to tailor the design, composition, and construction of organoids according to the intended purpose of the study. Here, I present an in vitro experimental platform for the organoid culture to design and control microenvironment. The results in collective cell behaviour under the controlled environment provides us the insight how ECM design is important to control morphology. Then, the simple cube device, which comprises a polycarbonate frame with rigid agarose walls and an inner ECM hydrogel, can be used to (i) control the spatial distribution of cells by employing 3D-printed carbohydrate moulds to create cell seeding pockets in the ECM hydrogel, (ii) design tissues with localized ECM by isolating ECM hydrogels of varying the composition or stiffness in separate compartments, (iii) facilitate integration with microfluidics to generate the concentration gradient of morphogens to direct cell growth and differentiation, and (iv) assemble multi-CUBE with organoids or tissues to express tissue-tissue interactions. Varieties of applications to locally differentiate from iPSCs as well as control branch pattern formations of bronchial cells have been achieved. The platform does not require complicated external system such as pump and laser. Thus, it can be easily installed in most of lab environment to accelerate the research for organoid developments while it is envisioned that increasingly sophisticated and interconnected organoids will be achievable.

Amyloids are insoluble, fibrilar protein aggregates, commonly known for their role in the development of neurodegenerative disorders. However, more recent studies show that such structures can be utilized by a variety of organisms to perform physiological functions including biofilm formation, hormone storage and signaling. Furthermore, it was shown that both functional and pathological amyloids can interact in a number of ways. Such interactions can lead to a significant increase in aggregation rates or inhibition of fibril formation. Despite the importance of amyloids, our understanding of processes governing their sefl assembly is still quite limited. The available experimental methods of studying amyloids are expensive and time-consuming. To overcome this problem several computational methods were proposed, but their capabilities are still limited. During my PhD, I developed a new method for the identification of aggregation-prone regions in proteins - PATH (Prediction of Amyloidogenicity by THreading) as well as a method for amyloid interaction predictions - PACT (Prediction of Amyloid Cross-Interactions by Threading). I also developed approaches for the identification of amyloids in proteome-wide studies. Finally, I aimed at investigating the possible interactions between functional amyloids produced by the gut microbiome and human disease-related amyloids.

During development, simple epithelial sheets give rise to a plethora of tissues with complex forms and specialized functions through regulated and robust tissue choreography. Mechanical forces, influenced by the microenvironment and generated within the morphing tissue, play a crucial role in shaping tissues at precise locations and time points to ensure proper form. Meanwhile, epithelial decision-making processes contribute to tissue patterning by accurately specifying correct cell types within the overall cellular structure, giving rise to a functional tissue. To robustly and successfully create form and function, tissue mechanics and epithelial decision-making must be tightly interlinked. I will discuss, how we use engineering approaches to deconvolve the role of mechanical forces on morphogenesis and cell patterning in in-vitro human pluripotent stem cell (hPSC) derived model systems. Specifically, I will discuss how mechanical forces instruct cell patterning and symmetry breaking in human neural tube organoids using a series of global and local actuation (mechanical stimulation) devices coupled with a single cell transcriptomics atlas. Furthermore, we will delve into the significance of the extracellular matrix (ECM) as a dynamic mechanical landscape during early development. In particular, I will discuss my recent work on the reciprocal relation between cell-driven ECM flow and epithelial morphogenesis. Through this study, I show that symmetry breaking events at sites of morphogenesis are necessary to drive and guide ECM flow, while ECM flow, in turn, is necessary to maintain morphogenesis. I will also discuss the in-silico modelling of this reciprocal relation and present the insights gained from single-cell transcriptomic analyses that reveals how ECM flow gives rise to a primitive streak-like phenotype in this pre-/early streak in-vitro gastrulation model

In vertebrates, the formation and maintenance of complex three-dimensional shapes in epithelia drives several important developmental processes. In addition, a collection of recent discoveries both in vivo and in vitro suggests that the collective mechanical response of a tissue (its fluidity or rheology) helps to control morphogenetic events. Therefore, there is a need to develop fully 3D biophysical models for epithelia that can predict or validate how global tissue mechanics impacts the structure and function of epithelia. In this talk, I will discuss several recent projects by our group and others to develop such models for specific model systems, including the left-right organizer in zebrafish and stratification and placode formation in mouse skin, where there are interesting interactions between cell shapes, epithelial architecture, and tissue fluidity. I will highlight how an interplay between models and experiments can help to drive a better mechanistic understanding of the processes that drive 3D tissue structure and function, especially when multiple mechanisms are operating at the same time.

The mission of the Hnisz laboratory is to discover principles that underlie control of transcriptional programs during development and disease. We recently proposed a model that transcriptional regulatory proteins form nuclear condensates that play important roles in the control of cell identity of mammalian cells. The central theme of the lab is to use the transcriptional condensate model to solve major outstanding problems in transcription- developmental- and disease biology. I will describe new insights into the molecular basis of condensate formation, regulatory functions of transcriptional condensates, and their alterations in genetic diseases in humans.

Active forces in biological systems define the interactions between single molecules, growing cells and developing tissues. Over the last three decades atomic force microscopy (AFM) has become an indispensable tool for characterisation of biological systems, as it enables the structural and mechanical characterization of samples at near native/physiological conditions. Nevertheless, biological systems are typically very heterogeneous and dynamic, which could pose challenges during cell surface analysis or identification of molecules. We will discuss how correlative BioAFM is used in life science microscopy core facilities nowadays to understand the structural, functional, chemical, mechanical, and dynamic complexities of biological systems. We will introduce and give examples of how the combination of AFM imaging with advanced super-resolution optics leverages the advantages of immunolabelling techniques for truly correlative microscopy. Through an innovative sample stage design, we will show how a wide choice of scanners, together with optical tiling and multi-region AFM probing, enables multiparametric mechanical characterization of soft samples over a large area and provides additional optical data sets.

Apico-basal polarity is an important determinant of epithelium structure and orientation. Polarity is perturbed or lost in various epithelial cancers, often at the start of metastasis. Such polarity disturbance could be in part, due to the changes in the extracellular matrix (ECM) in the tumor microenvironment, which undergoes biomechanical modifications. Here we study the impact of the extracellular matrix on the apico-basal polarity of patient-derived gastric cancer organoids. We show that normal gastric organoids require interaction with the ECM in order to maintain normal polarity. Notably, they dramatically switch their polarity when the integrin &#946;1 signaling pathway is inhibited. On the contrary, we find that cancer organoids inherently display heterogeneous phenotypes of polarity disturbance. We further discover that at least one gastric cancer line does not fully depend on the interaction with the ECM for maintaining polarity. We attempt to sub-clone this line and find potential evidence of genetic heterogeneity leading to such ECM independence

Mechanical processes are central to diverse cellular functions but can also be sources of cellular stress leading to aging phenotypes. My lab currently investigates three problems related to cell mechanics and stress: 1) how intracellular fluid dynamics coupled with cytoskeletal forces drive early mammalian development and reproductive aging; 2) how stress-induced protein aggregation and subsequent disaggregation are orchestrated by and affect organelles such as mitochondria and ER; and 3) how genome stability is affected by physical stress and how this contribute to cellular adaptation and cancer evolution. I will present a combination of recent findings in the first two areas of our research.

This talk will summarize three main techniques we developed in our Bioinformatics Lab in Catania. 1) MITHrIL: This system uses the concept of a metapathway, a mechanistic model of the cell, merging KEGG and REACTOME extended with microRNA and TF in a multi-relational network. MITHrIL takes as input the Log-Fold-Changes of differentially expressed biological elements (genes, metabolites, miRNAs). It will produce a quantitative prediction of each gene and pathway perturbation with its statistical significance. 2) PHENSIM: This system uses the MITHrIL model to simulate phenotypes. Phensim takes as input user-specified positively and negatively perturbed elements (genes, metabolites, miRNAs) and non-expressed genes in the cellular context (i.e., tissue, cell line) and will produce a qualitative prediction of gene and pathway alteration (positive, negative, or null) with its statistical significance. 3) NETME: This algorithm builds a knowledge graph starting from bio-medical literature given as input by the user. Papers can be obtained directly by querying PubMed or as PDF documents. The knowledge graph built by NETME summarizes the knowledge contained in those documents. We will show examples of these techniques for drug suggestion in infection and other diseases for pandemic first-aid intervention and precision medicine. Other applications will be briefly discussed, such as gene knock-out and knock-down simulation and extra-cellular exosome functional prediction. Finally, possible future extensions to single-cell and cell-cell communications will be outlined.

During pre-implantation development, the mammalian embryo forms the blastocyst. The architecture of the blastocyst is essential to the specification of the first mammalian lineages and to the implantation of the embryo. Consisting of an epithelium enveloping a fluid-filled lumen and the inner cell mass, the blastocyst is sculpted by a succession of morphogenetic events. These deformations result from the changes in the forces and mechanical properties of the tissue composing the embryo. Combining microscopy, image analysis, biophysical tools and genetics, we study the mechanical and cellular changes leading to the formation of the blastocyst.

Large pools of epithelial and interstitial stem cells define the well-known features of Hydra polyps such as their enormous capacity for regeneration, permanent growth and longevity. Multi-functional epithelial stem cells also shape Hydra’s body and regulate transport of ions, molecules and water. Two topics will be discussed: (1) the role of Myc factors in self renewal and differentiation, and (2) the role of Claudin cell contact proteins in epithelial integrity and regeneration.

The conserved yeast E3 ubiquitin ligase Bre1 (human RNF20/40) and its partner, the E2 ubiquitin conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle. Although processive ubiquitination might, in principle, arise from Bre1 and Rad6 travelling with RNA polymerase II, the mechanism of H2B ubiquitination across genic nucleosomes has remained unclear. We suggest that layered condensates of histone-modifying enzymes generate chromatin-associated ‘reaction chambers’, with augmented catalytic activity on gene bodies. I will present our recent progress on this topic integrating single-molecule with genome-wide studies of condensate behavior and functional genetic analyses.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. We are particularly interested in the role of fibroblasts in mammary epithelial morphogenesis. Using single-cell RNA sequencing and spatial mapping, we discovered a yet undescribed fibroblast subpopulation specific to the microenvironment of actively growing and branching epithelium. I will present our insights into regulation and function of this specific subpopulation, which in some aspects resembles cancer-associated fibroblasts.

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of post-mitotic neurons and glia. The talk will discuss the link between developmental lineage and cell type diversity, which is still an open question in the field. We used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence in all major cell classes of the mouse forebrain and found several types of GABAergic neurons that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis at cell cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor level. The talk will also discuss a mechanism by which selective enhancer activation is critical for establishing GABAergic projection neuron identity and potential pathological mechanisms.

As cells proceed through development, information contained in the genome is expressed in a context-dependent manner. This must be regulated precisely in both space and time to generate patterns of gene expression that set-up the spatial coordinates of tissue and organ primordia that build the embryo. Our current understanding of pattern formation relies on the concept of positional information, the idea that cells receive instructive signals that impart a spatial coordinate system to generate pattern. While this model works very well in static cell populations with minimal cell rearrangement, it becomes challenging when considering dynamic morphogenetic processes such as gastrulation. Furthermore, pattern formation in gastrulation is highly flexible to alterations in the size, scale and spatial rearrangement of cells in both experimental and evolutionary situations. Our work seeks to provide illustrations of two concepts that will help resolve these long-standing problems of pattern regulation, evolvability and self-organisation. Firstly, downward causation emphasises the role that multi-tissue interactions play in relaying information from changes at the organ and organism level to the regulation of gene regulatory networks (GRNs) at the cell level. Secondly, pattern emergence considers how extracellular signals act to control the dynamics of autonomous GRN activity, rather than as instructive signals to direct cell fate transitions. In this sense, we propose that pattern formation should not be seen as a downstream output of organisers and their responding tissues, but rather as an emergent property of their dynamic interaction.

One of the tenets of molecular biology is that dynamical transitions between three dimensional structures largely determine the function of proteins. Therefore, it seems only natural that evolutionary analysis of proteins, presently based on their primary sequence, needs to shift its focus towardsprotein function, as assessed by corresponding structural transitions. We will show how to adapt the formalism of finite strain analysis, developed in condensed matter physics and engineering, and apply it to structural transitions of proteins. As a case study, we will focus on the ATP synthase, for which our Protein Strain Analysis (PSA) provides a strain distribution on the protein structure associated with functional transitions. By analyzing the strain patterns for ATP synthases across different species, we show that they are evolutionarily conserved for the same functional transition. This observed strain conservation across evolutionary distant species indicates that this quantity should be essential in future structure-based evolutionary studies of protein function.

Enhancers can regulate their target promoters over large genomic distances for which spatial proximity between these elements is assumed to be necessary. Topologically Associating Domains (TADs) are thought to provide a spatial environment in the nucleus, harboring multiple enhancers and are thus related to gene regulation. However, to which degree TADs contribute to gene regulation and how enhancer position within the TAD affects its function in vivo is not known. I will present work that uses genome-engineering to generate a series of mouse lines with alleles designed to test how important TADs are for gene regulation. At well-characterized developmental loci, we either re-arrange TAD boundaries or reposition individual enhancers and analyze the effects on 3D chromatin structure, gene expression and mouse phenotype in vivo. Thereby we can show that TADs provide a supportive environment for enhancer function but also point to our limited understanding of the interplay between regulatory elements.