Physical Biology Circle Meeting

Interactions between liquids and surfaces generate forces that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water, modulation of the material properties of spider silk, and self-assembly of microstructures. Recent studies have shown that cells assemble biomolecular condensates via phase separation. In the nucleus, these condensates are thought to drive transcription, heterochromatin formation, nucleolus assembly, and DNA repair. In this talk, I will describe experiments and theory where we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism could act as a possible general regulatory principle for chromatin organization.

The development of an organism is characterized by a series of cellular fate transitions where cells become increasingly specialized. For many animal cells, fate transitions are accompanied by shape changes and there are strong indications of coupling between cell shape and fate. Here, we present a pipeline to quantify and analyse cell shapes as cells undergo the epithelial-mesenchymal transition (EMT). We will present how the morphological features of a cell can be quantified and analysed with the help of dimensional reduction methods. We then apply our analysis pipeline to investigate the coupling between cell shape and fate during EMT of MDCK cells. We find that cell morphology is closely associated with their state: While epithelial cells display spherical shapes, mesenchymal cells undergo spreading. After defining the distinct cellular shapes corresponding to cell states, we study how exactly the morphological features of a cell evolve during EMT. To this aim, we investigate cell trajectories of morphological features in a low-dimensional space and describe the time evolution of cellular features as a stochastic process. By integrating morphometric analysis into studies of cell fate changes, we aim to better understand the crosstalk between cellular fate and shape changes.

What determines the fate of a stem cell? In adult skin tissue, stem cells - which are responsible for repair and renewal - are confined to the bottom-most basal layer (attached to a basement membrane with high rigidity) and loose their ‘stemness’ irreversibly when it extrudes/leaves the basal layer into the supra-basal layer above. While several cell-intrinsic biochemical markers have been discovered to predict stem cell potential, we explore a complementary hypothesis to this question with the help of tissue biomechanics, that can take into account the cell extrinsic mechano-chemical changes in the environment. We use a fully 3D vertex-like model to study the differentiation propensity in terms of cell extrusion dynamics, and its dependence on global mechanical properties like- tissue fluidity and basement tension. Surprisingly, tissue surface tension in such confluent tissues can induce unique interfacial behaviour like- cell shape elongation and topological pinning- that can make perhaps make cells extrusion much costlier, possibly resulting in a delayed onset of differentiation. We investigate the extrusion dynamics across the fluid-solid transition and will conclude by discussing the phenomenon with the help of a simple 2D toy model.

The yeast mating-pheromone pathway is the best understood MAP-kinase signal transduction pathway in eukaryotes. The MATa and MATα mating-types exchange pheromones to trigger expression of mating genes and find each other in space through chemotropism. The interplay between pheromone production and degradation ultimately determines mating behaviour. This process has been extensively studied using artificially generated gradients of purified pheromone. However, the spatial pheromone landscape that responding cells experience during mating is that of multiple “point” sources of overlapping gradients that further actively modify their position in space and are subject to active degradation by a specific protease, the “barrier” protein Bar1. Here we present the development of optogenetic yeast strains, where these processes can be controlled with spatiotemporal precision using light. These tools can be used to study the mating process by generating sensory input landscapes at will from their native source cells, and therefore provide controlled yet realistic quantitative characterization.

The continuous progress in imaging and computer modelling have increased our understanding of morphogenetic processes at different scales, from organs up to entire organisms. However, in the case of complex animal models (e.g. mouse embryogenesis), is not yet entirely possible to observe in real time the full growth of a developing embryo. Consequently, the current 3D data availability in these cases, even if extensive and detailed, provides only a characterisation of development at discrete moments in time, through single snapshots. To fill this gap, we set up a computer-based approach to describe the evolution in time and space of developmental stages from 3D volumetric images. Specifically, we represent each data into the spherical harmonics space, and we reconstruct the volumes using the values of the spherical harmonics coefficients interpolated in time (over the developmental stages). As a result, the reconstruction describes a continuous and smooth changing shape over space and time. We tested this approach using two different data sets: (1) mouse limb buds and (2) mouse hearts. (1) ~100 optical projection tomography (OPT) of mouse limbs were used and the result represents the 4D growth of an ideal limb which considers the common characteristics and features of all the limbs in the data set. We are recreating the growing process starting from E10 (i.e. 10 days after conception) when the limb bud is just a small bump of tissue and finishing at E12.5 when the limb bud already shows a distinctive “paddle” shape. This approach proved to be robust even in the case of mouse hearts (2) where only a limited number of sample (~30) were used. Also in this case we were able to create a continuous time-course describing the heart development of a mouse starting from 10 to 28.5 somites. This approach, able to combine the complexity of different arbitrary shapes over space and time, not only provides a quantitative basis for validating predictive models, but it also increases our understanding of morphogenetic processes from a purely geometrical point of view.

Many living systems need to react to input signals and produce a corresponding output. Using information theory, we can quantify the amount of information the system's output contains about the corresponding input signal by computing the mutual information between input and output signals. In biochemical signaling, information is frequently encoded in the full temporal dynamics of the input or the output. Hence, we need to consider whole input/output trajectories for the computation of the mutual information. However, analytical results for mutual information between trajectories are only available for simple Gaussian systems. But also the conventional computational techniques to calculate the mutual information are infeasible for high-dimensional objects such as trajectories. We present a novel Monte Carlo technique to compute the mutual information between input and output trajectories for the large class of biochemical systems that are described by a chemical master equation. The core idea is to use the chemical master equation to evaluate the exact probability of individual output trajectories for a given input trajectory. Based on this idea we derive an exact Monte Carlo estimate to compute the corresponding mutual information. The required stochastic trajectories are generated using Gillespie's algorithm. For a simple gene expression model, we benchmark our results against analytical approximations of the mutual information. To demonstrate full power of our algorithm we then apply it to a realistic model of bacterial chemotaxis, a system consisting of 173 reactions due to the large number of receptor states, and show that our method is capable of computing the information rate of such large and complex systems.

Mechanics has been a central focus of physical biology in the past decade. In comparison, the osmotic and electric properties of cells are less understood. Here we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spread, migrate or are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechano-senstive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.

In response to a wound, cells inside a tissue must organise a collective migration in order to close the gap. In epithelial monolayers this behaviour is accompanied by wave patterns in the motion of cells as well as strikingly similar patterns in their chemical ERK activity. We therefore ask how cell mechanics is coupled to chemical state, whether feedbacks in this coupling can lead to pattern formation and what role the patterns play in coordinating cell migration. We begin by formulating a simple mechano-chemical model, coupling a passive elastic tissue to active cellular stresses and the mechano-sensitive ERK pathway, before fully parameterising it through mechanical and optogenetic perturbation experiments. The model successfully predicts patterning, explaining its mechano-chemical origin and providing quantitative agreement in terms of the period and wavelength of the observed waves as well as their correlations. We go on to propose a mechanism coupling the patterns to cell propulsion and demonstrate that it robustly induces motion in the desired direction. We also determine the theoretically optimal wave for inducing the strongest migration response and find that it agrees well with experimentally measured values. In this way the work goes beyond description of the mechano-chemical phenomenon in order to ask questions regarding the design principles of the system.

We discuss the stochastic trajectories of single molecules in a phase-separated liquid, when a dense and a dilute phase coexist. Starting from a continuum theory of macroscopic phase separation we derive a stochastic Langevin equation for molecular trajectories that takes into account thermal fluctuations. We find that molecular trajectories can be described as diffusion with drift in an effective potential, which has a steep gradient at phase boundaries. We discuss how the physics of phase coexistence affects the statistics of molecular trajectories and in particular the statistics of displacements of molecules crossing a phase boundary. At thermodynamic equilibrium detailed balance imposes that the distributions of displacements crossing the phase boundary from the dense or from the dilute phase are the same. Our theory can be used to infer key phase separation parameters from the statistics of single-molecule trajectories. For simple Brownian motion, there is no drift in the presence of a concentration gradient. We show that interactions in the fluid give rise to an average drift velocity in concentration gradients. Interestingly, under non-equilibrium conditions, single molecules tend to drift uphill the concentration gradient. Thus, our work bridges between single-molecule dynamics and collective dynamics at macroscopic scales and provides a framework to study single-molecule dynamics in phase-separating systems.

Deformations of epithelial tissues in 3D, which are crucial for embryonic development or in vitro organoid growth, can result from active forces generated within the cytoskeleton of the epithelial cells. How the interplay of local differential tensions with tissue geometry and with external forces results in tissue-scale morphogenesis remains an open question. Here, we describe epithelial sheets as active viscoelastic surfaces and study their deformation under prescribed internal tensions and bending moments. In addition to isotropic effects, we take into account nematic alignment in the plane of the tissue, which gives rise to shape-dependent active nematic tensions and torques. We present phase diagrams of the mechanical equilibrium shapes of pre-patterned closed shells and explore their dynamical deformations. Our results show that a combination of nematic alignment and tension gradients is sufficient to reproduce the basic building blocks of epithelial morphogenesis, including fold formation, budding, and tubulation.

Despite the absence of a membrane-enclosed nucleus, the bacterial DNA is typically condensed into a compact body - the nucleoid. This compaction influences the localization and dynamics of many cellular processes including transcription, translation, and cell division. We have developed a theoretical model that takes into account steric interactions among the components of the Escherichia coli transcriptional-translational machinery (TTM) and out-of-equilibrium effects of messenger RNA (mRNA) transcription, translation, and degradation, in order to explain many observed features of the nucleoid. We show that steric effects, due to the different molecular shapes of the TTM components, can drive equilibrium phase separation of the DNA, explaining the formation and size of the nucleoid. In addition, we argue that the observed positioning of the nucleoid at midcell can be due to the out-of-equilibrium process of mRNA synthesis and degradation: mRNAs apply an osmotic pressure on both sides of the nucleoid, localizing it to midcell. We demonstrate that, as the cell grows, the production of these mRNAs can explain the nucleoid splitting into two lobes, and their well-known positioning to 1/4 and 3/4 positions on the long cell axis. Overall, our study suggests that steric interactions and out-of-equilibrium effects of the TTM are key drivers of the internal spatial organization of bacterial cells.

Multicellular aggregates are known to exhibit liquid-like properties. The fusion process of two cell aggregates is commonly studied as the coalescence of two viscous drops. However, tissues are complex materials, which usually exhibit viscoelastic behaviour. It is known that elastic effects can prevent the complete fusion of two drops, a phenomenon known as arrested coalescence. Here we show that this phenomenon can be exploited to infer the mechanical properties of tissues. By fitting the dynamics of the fusion process to a viscoelastic model and combining this method with nanoindentation, it is possible to obtain a complete mechanical characterisation of the aggregates. We illustrate our method by studying the fusion dynamics of aggregates of mouse embryonic stem cells. Additionally, agent-based simulations suggest that arrested coalescence can be found in the vicinity of an unjamming phase transition. Our work provides a simple in vitro method to characterize the mechanical properties of 3D multicellular aggregates and sheds light on the impact of cellular activity on tissue mechanics.

While many tissues fold in vivo in a highly reproducible and robust way, epithelial folds remain difficult to reproduce in vitro, so that the effects and underlying mechanisms of local curvature on the epithelial tissue remains unclear. Here, we photoreticulated polyacrylamide hydrogels though an optical photomask to create corrugated hydrogels with isotropic wavy patterns, allowed us to show that concave and convex curvatures affect cellular and nuclear shape. By culturing MDCK epithelial cells at confluency on corrugated hydrogels, we showed that the substrate curvature leads to thicker epithelial zones in the valleys and thinner ones on the crest, as well as corresponding density, which can be generically explained by a simple 2D vertex model, leading us to hypothesize that curvature sensing could arise from resulting density/thickness changes. Additionally, positive and negative local curvatures lead to significant modulations of the nuclear morphology and positioning, which can also be well-explained by an extension of vertex models taking into account membrane-nucleus interactions, where thickness/density modulation generically translate into the corresponding changes in nuclear aspect ratio and position, as seen in the data. Consequently, we find that the spatial distribution of Yes associated proteins (YAP), the main transcriptional effector of the Hippo signaling pathway, is modulated in folded epithelial tissues according to the resulting thickness modulation, an effect that disappears at high cell density. Finally, we showed that these deformations are also associated with changes of A-type and B-type lamin expression, significant chromatin condensation and to lower cell proliferation rate. These findings show that active cell mechanics and nuclear mechanoadaptation are key players of the mechanistic regulation of epithelial monolayers to substrate curvature, with potential application for a number of in vivo situations.

Lipid and protein sorting are crucial processes that maintain unique biophysical and biological properties of different organelles. Nevertheless, little is known about mechanisms behind lipid sorting. Sphingomyelin (SM) is synthesized at the trans Golgi network (TGN) and transported to the plasma membrane (PM) via an uncharacterized pathway. SM enrichment in transport carriers cannot be explained by a curvature-based mechanism of lipid sorting due to its property to form stiff membranes. A mechanism of protein-mediated lipid sorting has been proposed, taking advantage of protein’s affinity for curved membranes and specific lipids. One of the proteins enriched in the same secretory pathway is caveolin, which shuttles between the Golgi and the PM where it forms cup-shape, SM-cholesterol-enriched domains called caveolae. The assembly process is initiated by the export of caveolin-enriched vesicles from the Golgi but little is known about this step except from a critical role of lipids in caveolin oligomerization. The goal of this project is to determine the role of caveolin in SM trafficking from the TGN to the PM and to decipher the molecular mechanisms behind caveolin assembly. Our in cellulo studies show significant changes in SM distribution in cells devoid of caveolin comparing to wt, indicating that caveolin play a role in SM trafficking. Our recent findings obtained with RUSH technology, which enables synchronized release and trafficking of desired molecules within the cell, demonstrate significantly slowed down SM Golgi-exit in the absence of caveolin. Moreover, SM trafficking is redirected towards lysosomes proving that indeed caveolin is involved in proper SM transport and distribution within the cell. Simultaneously we used a bottom-up approach and for the first time we managed to reconstitute caveolin 1 (Cav1) into small unilamellar vesicles and analysed their structure with cryo-EM, in context of different lipid composition. Furthermore, we established a novel in vitro system in which we reconstituted Cav1-SM complex in giant unilamellar vesicles (GUVs). Protein/lipid sorting was analysed by tube pulling approach with the use of micropipette aspiration coupled with optical tweezers. Acknowledgments: This work was supported by the Labex CeltisPhybio, Polish Ministry of Science and Higher Education – program ‘Mobility Plus’

Transition from Interphase chromatin to mitotic chromosome formation is one of the most dramatic changes during cell cycle. Among many, SMC protein complex- ‘Condensin’ have been attributed to the main factor responsible for this event. Recently through an in vitro study, it has been shown that condensin complex from budding yeast make DNA loops which grows over time and a mechanism of condensin mediated loop extrusion has been proposed as a way of DNA compaction in vivo. Here we characterize the activity of condensin complex from a thermophilic yeast species Chaetomium thermophilum (Ct). Condensin holo-complex was purified and confirmed by mass spectrometry. Rotary shadowing followed by electron microscopy imaging was performed to observe intact condensin complex and preliminary data suggested that condensin can compact DNA in an ATP dependent manner. The condensin complex also has DNA stimulated ATPase activity as shown by ATPase assay. To generalize the loop extrusion mechanism, we imaged lambda DNA with Condensin protein complex and observed DNA loop extrusion in real time at single molecule resolution. This process in strictly ATP dependent and the process asymmetric. When one of the subunits, previously noted as ‘DNA anchor site’ was deleted, to our surprise we not only observed loop extrusion, this ‘anchor-less’ protein complex can now change directions during loop extrusion, making condensin complex a symmetric extruder from an asymmetric one. Furthermore, we determined that the so called ‘anchor-less’ protein complex is robust to external tension and stall force and a more efficient extruder. Our data brings a better understanding about the molecular process of lop extrusion and elucidates chromatin dynamics.

One of the key characteristics of life is the ability of cells to divide. During division, the replicated genetic material, in the form of long DNA polymers, has to be properly spatially segregated and apportioned to the nascent daughter cells. It has been suggested that entropy could be the driver for chromosome segregation, based on polymer theory and simulation. In recent years, the prerequisites for and characteristics of entropic segregation have been studied in more detail, mostly for elongated E.Coli-like cell shapes, which turn out to be especially suitable for this process. We aim to develop this principle into a working protocol that could function within the design parameters of a future autonomously dividing synthetic cell. Specifically, we work in the context of the Dutch Building a Synthetic Cell (BaSyC) consortium, which has a bottom-up approach to making minimal life and would thus welcome a simple, robust, protein-free method of segregation. We explore the options for reliable segregation in this context with course grained molecular dynamics simulations and phenomenological polymer theory. The BaSyC cell will likely be a spherical vesicle, which is constricted into a dumbbell shape during division. This shape poses specific challenges to the entropic segregation process. The required lateral confinement is weak in most places, but strong in the neck region, which acts as a barrier. The longitudinal space for the chromosomes to escape into is limited. The negative impact on the segregation speed and reliability depends on parameter choices, for the container as well as the chromosomes. For instance, adding topological constraints like loops increases the repulsion between polymers and leads to reliable segregation even in otherwise unfavourable container shapes or chromosome volume fractions. Together, our findings so far provide guidelines for the selection of the BaSyC container, chromosome and additives like SMCs and crowding agents. In turn, the process of building a synthetic cell and choosing its properties may offer insights about minimal biological cells too.

Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. Here we map the three-dimensional cell-ECM and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. These force patterns co-evolve with fate specification to progressively shape a mature compartmentalized epithelium. The size of the stem cell compartment depends on ECM stiffness and on endogenous cellular forces. A 3D vertex model shows that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we show that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal crypt.

Adhesion is a ubiquitous process for virtually all cells from those that form together tissues and organs of multicellular organisms to single immune cells that migrate adhering to the extracellular matrix in search of pathogenic cells or tiny bacteria that adhere to their host cell’ membrane in order to infect them. Cells adhere to substrates or other cells not only to maintain physical cohesion, but also to receive information on their microenvironment. Adhesion is thus a platform for communication (chemical and mechanical) between a cell and a substrate or another cell. In this work, we are interested in integrin-mediated adhesion. The adhesion proteins are the main players of this mechano-chemical communication. During cell adhesion they assemble in clusters and in a process called mechanotransduction convert mechanical stimuli into biochemical signals and transmit them inside the cell. Although cell adhesion on rigid substrates is very well studied, much about adhesion on soft fluid substrates like membranes remains unknown. Here we have studied the formation and evolution of integrin clusters in cells adhering on fluid lipid bilayers functionalized with cell ligands of different affinities (RGD, Invasin) at the early stage of cell adhesion. Ligand affinity to integrins appears to be very important in integrin activation and consequent formation of adhesion clusters. We have used advanced confocal microscopy to demonstrate that during adhesion cells are able to deform fluid bilayers by pulling membrane tethers from them. Additionally, we have observed the recruitment of the proteins that are associated with mature adhesions to integrin clusters like zyxin and VASP, which suggest the presence of mechanotransduction in integrin clusters on fluid substrates. Moreover, we have used fluorescence calibration and custom-made image analysis routines to quantify integrin densities in adhesion clusters. We have found that integrin densities in clusters on Invasin are significantly higher than the ones on RGD reaching the densities reported for focal adhesions on glass. It suggests that ligand affinity is a critical parameter in integrin cluster growth. Integrin densities in clusters on Invasin increase in a non-linear step-like manner suggesting that the clusters grow by fusion.

Embryonic development offers many examples of emergent properties: organs form in a way that is difficult to predict by observing their individual elements, the cells but depends on interactions in the collective. This is however taking place in complex environments where organs are open systems submitted to external controls originating from other organs and the embryo environment. Organoids are three-dimensional cultures initiated from cells which have the ability to both duplicate and become differentiated into more specialized cell types. They offer a simplified setting mimicking some aspects of development (usually not all) where the organ-forming cells can be manipulated and monitored in an easier way, outside of the embryo to uncover principles of self-organization. To understand pancreas development, as a complement to in vivo investigations, we designed 3D culture conditions that enable the efficient duplication, differentiation and morphogenesis of dissociated mouse embryonic pancreatic progenitors. We will show examples illustrating that the collective trajectories of the cells depend on: (1) the initial conditions such as cell number and cell state (2) signals the cells exchange (3) the signals the cells receive from the environment, such as the culture media. We will also illustrate how organoids can be used to study the morphogenesis of organs and its control by an interplay between tissue mechanics, hydraulics and the material properties of the environment.

The maintenance of intact tissues relies on precise cellular decision-making despite strongly fluctuating extrinsic cues. These decisions involve processes on vastly different scales, from molecules to organelles and cells in tissues. How can cells manipulate the flow of information across these scales to perform biological function? Here, we show how the non-equilibrium interplay between molecular and organelle dynamics controls the propagation of time-dependent signals to a response on the cellular scale. Specifically, by mapping the time evolution of organelle-associated signalling molecules to the physics of Josephson junction arrays, we demonstrate the emergence of a single, collective degree of freedom. We show that this degree of freedom exhibits intriguing dynamical behavior on different temporal scales leading to a kinetic low-pass filter allowing cells to distinguish between fast noise fluctuations and slow biological signals. We demonstrate our findings in the context of the metabolic regulation of cell death via the interplay of Bax protein dynamics with rapid mitochondrial fusion and fission and find an order of magnitude effect on the error rate of the cell death decision. We experimentally validate our conclusions by temporal apoptosis assays. Our work displays a paradigmatic example how biological function relies on the non-equilibrium integration of processes on different spatial scales to control and respond to fluctuations.

Efficient immune-responses require migrating leukocytes to be in the right place at the right time. When crawling through the body amoeboid leukocytes must traverse complex three-dimensional tissue-landscapes obstructed by extracellular matrix and other cells, raising the question how motile cells adapt to mechanical loads to overcome these obstacles. Here we reveal the spatio-temporal configuration of cortical actin-networks rendering amoeboid cells mechanosensitive in three-dimensions, independent of adhesive interactions with the microenvironment. In response to compression, Wiskott-Aldrich syndrom protein (WASp) assembles into dot-like structures which act as nucleation sites for actin foci forming bumps that in turn brace against the external load. High precision targeting of WASp to objects as delicate as collagen fibers allows the cell to locally and instantaneously deform its viscoelastic surrounding in order to generate space for forward locomotion. Such pushing forces are essential for fast and directed, amoeboid leukocyte migration in fibrous and cell-packed tissues such as skin and lymph nodes.

The nucleolus is a multi-phase nuclear condensate in the eukaryotic nucleus that contains rDNA and is surrounded by perinucleolar chromatin. It is composed of three canonical subcompartments: the fibrillar centre (FC) and the dense fibrillar component (DFC) and the granular component (GC), which are essential for ribosome biogenesis. Recent studies suggest that the distinct nucleolar subcompartments arise from different biophysical properties of the individual phases such as droplet surface tension. Upon DNA damage the nucleolar structure is reorganized as a consequence of suppressed RNA polymerase I (Pol I) transcription and the FC and DFC compartments form specific structures at the nucleolar periphery called nucleolar caps. The underlying mechanisms of this reorganization process remain poorly understood. To shed insight into the reorganization process, we performed 4-D live-cell imaging of the three subcompartments upon Actinomycin D (Act D) treatment, which inhibits Pol I transcription. By quantitative image analysis we uncovered a two-step process of nucleolar cap formation: First, we detected rounding of the GC and at the same time rapid fusion events of individual FC and DFC foci inside the GC resulting in a rapid decrease of the number of FC and DFC foci. Subsequently, the few remaining foci moved from the inner GC region to the nucleolar periphery and formed nucleolar caps. Unexpectedly, when we labeled Ki-67 and DNA, we found that both were rapidly excluded from the nucleolar interior in the first phase. Since Ki-67 depleted cells showed faster DFC coalescence and migration to the GC surface compared to the control cells, we hypothesize that Ki-67 and/or DNA suppresses the nucleolar reorganization caused by Pol I inhibition. Our study not only provides a dynamic spatiotemporal map of the nucleolar reorganization upon Pol I inhibition but also gives first mechanistic insights into the reorganization process.

The cell surface is an essential mechanical system in the context of cell shape changes. It is composed of two layers, the plasma membrane and the underlying actomyosin cortex, which are physically connected by specialized linker proteins that mediate the so-called membrane to cortex attachment (MCA). The impact of MCA on cellular surface mechanics is the least understood so far, partly due the lack of available tools to experimentally perturb MCA in the past. Here, we have developed a new toolkit of artificial, signaling inert membrane-to-cortex linkers, based on previous work in our lab, in order to selectively manipulate MCA. This approach allows us to modulate specific linker properties, such as density or length and decipher its effects on cell surface mechanics We found that an increase in MCA leads to a softening of the cell surface in 3T3 fibroblasts and mESC, measured by atomic force nano indentation, suggesting that changes in MCA affect cell cortex mechanics. Specific measurements of mechanical properties of the cell cortex, such as cortical tension, elasticity and thickness confirmed a modulation upon an increase in MCA. Using our tools in combination with a perturbation of cortex-specific nucleators (Arp2/3 and formins) further suggested that MCA might control the cell cortex by regulating cortical architecture. Encouraged by our findings so far, we are aiming to elucidate the mechanism by which MCA can regulate cortical architecture and thus cortex mechanics to gain more insights in how the different layers of the cell surface work together, which will be key to understand both local and global control of cell form and function.

Morphogenesis depends crucially on the complex rheological properties of cell tissues and on their ability to maintain mechanical integrity while rearranging at long times. In this talk, I will discuss the rheology of polygonal cellular networks described by a vertex model in the presence of fluctuations. I will introduce a triangulation method to decompose shear into cell shape changes and cell rearrangements. Considering the steady-state stress under constant shear, the nonlinear shear-thinning behavior at all magnitudes of the fluctuations, and the even stronger nonlinear regime at lower values of the fluctuations will be explored. I will discuss how this nonlinear rheology can be successfully captured by a mean-field model that describes the tissue in terms of cell elongation and cell rearrangements. The role of anisotropic stresses will also be discussed.

In C. elegans, the insulin/IGF-1 pathway is responsible for mounting tailored responses to a broad range of external stresses. DAF-16/FOXO, the primary output of this pathway, translocates to the nucleus upon stress to induce gene expression. So far, it is assumed that the level of nuclear DAF-16 remains constant if stress conditions are unchanged. Surprisingly, when we visualized DAF-16 in individual L1 larvae exposed to constant stress, we instead observed stochastic pulses of nuclear translocation, with DAF-16 moving between the nucleus and cytoplasm in ~1hr pulses. Pulses were present both in DAF-16::GFP integrated transgenes and CRISPR knock-ins. The pulses were strikingly synchronized across the worm body, with cells from different tissues switching within minutes. Quantitative analysis revealed that DAF-16 pulse dynamics was specific to different types of stress: starvation resulted in pulses that resembled stochastic oscillations, osmotic shock yielded random pulses whose average durations increased with salt concentration while their amplitude remained constant, and temperature shock gave rise to a single high-amplitude pulse, followed by persistent DAF-16 nuclear localization. Overall, these results suggest that the worm could use differences in DAF-16 nuclear translocation dynamics to determine both magnitude and type of stress. We use mathematical modelling to provide a potential mechanism for DAF-16 pulse generation and their body-wide synchronization. Finally, we also observed pulsatile dynamics of the DAF-16 homolog FoxO in mammalian cells at low nutrient levels that activate insulin signaling. Overall, this indicates that DAF-16/FoxO translocation pulses are a general feature of insulin signaling.

Collective cell flows within tissues are central in biological processes such as morphogenesis, wound healing, or cancer progression. In vivo, cell behaviors are controlled by their surrounding environment such as oriented bundles of extra cellular matrix proteins. These fibrillar structures can be recapitulated in vitro with grooved substrates allowing to study the cell response to such cues by “contact guidance”. Up to now, contact guidance has been mostly studied on single cell migration. However, little is known about cell collective response to such cues. Here, we show that, when plated on subcellular grooves, a confluent monolayer of human bronchial epithelial cells (HBECs) spontaneously organizes into wide alternating polar lanes that migrate in antiparallel directions. These lanes can be few millimeters in length and coarsen with time reaching widths of several hundred micrometers before cells jam. Within a lane, cell displacements are described by a plug flow with a well-defined polarity at the tissue scale. Our results are well captured by a hydrodynamic description of an active polar fluid that undergoes a disordered-to-flocking transition favored by friction anisotropy via the damping of fluctuations in the transverse direction. More microscopically, particle based simulations encapsulating a friction anisotropy identify polarity-velocity coupling and excluded volume interactions as key ingredients of the laning transition.

Cellular errors lead to spontaneous cell death in the earliest stages of development. We identified that the embryonic surface epithelium formed in early blastula stages mediates efficient phagocytic clearance of apoptotic cells. We found that epithelial cells form two types of actin-based basal protrusions during target interactions: 1) phagocytic cups responsible for apoptotic target uptake and 2) “epithelial arms” that are able to exert mechanical pushing forces on targets. We here addressed how epithelial cells form two types of actin-based protrusions by studying the molecular and biophysical factors regulating phagocyte-target interaction dynamics. We showed that epithelial protrusion formation and phagocytic uptake is mediated by PS recognition at the apoptotic cell surface. Inhibition of Rac1, a downstream effector of PS receptor activation regulating actin dynamics, prevents both cup and arm formation. Pharmacological interference with actin polymerization regulators revealed that Arp2/3 is essential for both cup and arm formation. However, inhibition of Formins prevents phagocytosis but is not required for arm formation. Our data further suggest that a lack of Formin prevents the normal function of the phagocytic cup, while a lack of Arp2/3 inhibits its entire formation. We further identify a key role of cellular adhesion in regulating the efficiency of epithelial protrusion formation. These findings reveal distinct functions of actin regulators and adhesion properties in controlling epithelial protrusions plasticity and efficient phagocytic tissue clearance.

Several studies have demonstrated cancer cells are softer compared to their noncancer counterparts. Changes of cellular stiffness often relate to changes of the actin cortex. This cortex is a network of actin filaments and several associated proteins which is placed beneath the plasma membrane. Its stiffness is influenced by its molecular composition, actin concentration and its architecture. In vitro studies showed that the mesh hole area of the actin cortex directly influences stiffness. During the process of cancer cell metastasis cancer cells change in many aspects, one of them is their adhesive form: They are first embedded in tissue but then suspended once they are in circulation (e.g. in the blood vasculature). However, up till now it is not exactly known how the actin cortex changes during the cancer progression and how these changes impact the overall cell mechanics. Here, we study the actin cortex of cancer (Mcf-7) and noncancer (Mcf-10a) cells in an adhered and suspended state. We used scanning electron microscopy to quantify the actin cortex architecture and relate this to cortex stiffness measured by creep compliance atomic force microscopy. We found that the mesh hole size and stiffness correlate positively over the nucleus of adhered cells. In contrast, suspended cells show a highly ruffled and folded cortex which we define as the cortex microstructure. We found that the actin cortex microstructure rules over its nanostructure (e.g. mesh hole size). We show that in the end it is the microstructure which correlates with the cortex stiffness for cells in suspension.