Physics of Living MatterSymposium

14th Edition

2-3 September 2019

PRACTICAL INFORMATION

VenueDepartment of Applied Mathematics and Theoretical Physics (DAMPT)University of CambridgeCentre for Mathematical SciencesWilberforce RoadCambridge CB3 0WA

ProgrammeConference registration will open on Monday 2nd September at 8:30am with talks starting at 9:25am. On Tuesday 3rd sessions will begin at 9:30am. Full programme can be found in the next pages.

PostersPlease display your poster when you arrive at the venue. The poster session will take place on Monday afternoon at 17:35. The Poster prizes will be awarded on Tuesday afternoon. Posters will be on display throughout the conference.

Conference partyA Conference party will take place at St Catherine’s College, on Monday 2nd September at 19:00.St Catherine’s CollegeTrumpington StreetCambridge CB2 1RL

Organising CommitteeAndré BrownKevin ChalutKristian FranzeEwa Paluch

8:30 Registration

9:25 Welcome

Session 1

9:30 Alex Dunn | Stanford UniversityMolecular origins of symmetry breaking at cell-cell adhesion complexes

10:00 Andrew Stannard | King’s College LondonMechanically modulating the nuclear translocation of proteins

10:15 Dirk Benzinger | ETH ZurichPulsatile transcription factor regulation reduces cell-to-cell variability in gene expression

10:30 Ben Lehner | Centre for Genomic Regulation, BarcelonaSolving protein structures and understanding genetic interactions using deep mutagenesis

11:00 Coffee Break

11:40 Tribute to Suzanne Eaton:Ewa Paluch (University of Cambridge), IntroductionGuillaume Salbreux (Francis Crick Institute), Flows in the fly wing

12:25 Lunch

Session 3

14:00 Lukas Kapitein | Utrecht UniversityNavigating the neuronal cytoskeleton

14:30 Diana Fusco | University of CambridgeHow spatial growth constraints evolution: insights from microbes and phages

15:00 Maximilian Jakobs | University of CambridgeMicrotubule polarity in axons is sorted by a molecular gradient of dynactin

15:15 Colin Russell | Academic Medical Center, University of AmsterdamMultiscale evolution of influenza viruses

15:45 Coffee Break

Session 4

16:20 Assaf Zemel | Hebrew University JerusalemElastic interactions of cells and their role in cellular self-association

16:50 Nicoletta Petridou | Institute of Science and Technology, AustriaTissue morphogenesis at criticality

17:05 Michael Krieg| The Institute of Photonic Sciences, BarcelonaExploring force transmission pathways during touch and proprioception

17:35 Drinks & Poster Session

19:00 Dinner & Conference Party

Monday 2nd September

Session 5

9:30 Hyun Youk | Delft University of TechnologyCells reshape habitability of temperature by helping each other replicate

10:00 Serena Ding | Imperial College LondonShared behavioral mechanisms underlie C. elegans aggregation and swarming

10:15 Alison Sweeney | University of PennsylvanniaPhase transitions and photonics in cephalopods

10:45 Coffee Break

Session 6

11:15 Sally Lowell | University of EdinburghMis-shapes, Mistakes, Misfits and how to avoid them: how do pluripotent cells make the right decisions?

11:45 Henry De Belly | University College LondonMembrane tension regulates FGF driven fate choice in embryonic stem cells

12:00 Jared Toettcher | Princeton UniversityOptogenetic rescue of a developmental patterning mutant

12:30 Lunch

Session 7

13:45 Suliana Manley | École polytechnique fédérale de LausanneStochasticity and control in mitochondrial division

14:15 Rasha Rezk | University of CambridgeThe mechanical heterogeneity in human glioblastoma

14:30 Shahaf Armon |Weizmann Institute of ScienceFrom Contraction Waves to Active Resistance to Rupture – Tissues as Sheets of Relaxation Oscillators

14:45 Anne Grapin-Botton | Max Planck Institute of Molecular Cell Biology and GeneticsOrganoids towards an understanding of self-organization in organogenesis

15:15 Coffee Break

Session 8

15:45 Naama Barkai| Weizmann Institute of ScienceTBC

16:15 Walter Federle| University of CambridgeSlippery surfaces and sticky feet: Biomechanics of adhesion in insects

16:45 Closing Remarks & Poster Prizes

Tuesday 3rd September

Speaker Profiles

The Barkai group studies the design of bio-molecular networks, applying a systems biology approach that combines theory, computation data analysis and experimentations. We are interested in how cells process and integrate multiple internal and external signaling, and how this regulation has evolved.

Naama BarkaiWeizmann Institute of ScienceTuesday 3rd September, 15:45

Title TBC

The Dunn lab uses quantitative biophysical methods to understand the physical processes that control cellular function and embryonic development. We used fluorescent molecular tension sensors to measure forces transmitted at cell-cell junctions (Borghi et al. PNAS 2012), at single integrin complexes (Morimatsu et al., Nano Letters 2013, Chang et al. ACS Nano 2016), and within the neurons of living C. elegans (Krieg et al. Nat. Cell Biol. 2014). In related work, we used a single molecule optical tweezers assay to determine the probable molecular mechanism that underlies mechanotransduction at cellular adherens junctions (Buckley et al. Science 2014). More recently, we used this same assay to discover a novel physical mechanism by which the protein vinculin may assist in generating and maintaining long-range cytoskeletal organization in migrating cells (Huang et al. Science 2017). Work in zebrafish revealed a role for mechanical tension in guiding long-range cellular movements during early embryonic development (Chai et al. Biophys J. 2015), while experiments in C. elegans revealed the critical role for mechanical coupling between tension and torsion in maintaining the physical integrity of neurons (Krieg et al. eLife 2017). The results of these and other ongoing projects suggest deep commonalities in the mechanisms by which physical cues guide tissue morphogenesis.

Alex DunnStanford UniversityMonday 2nd September, 9:30

Molecular origins of symmetry breaking at cell-cell adhesion complexes

I am interested in the mechanics of animals and plants, and the adaptations they have evolved to survive in their environment. I have studied locomotion and the design of mechanical systems in insects, as well as functional surfaces, insect-plant interactions and the biophysics of animal attachment mechanisms.

Walter FederleUniversity of CambridgeTuesday 3rd September, 16:15

Slippery surfaces and sticky feet: Biomechanics of adhesion in insects

My group works on understanding how evolution allows populations of individuals to tackle different environmental challenges. In particular, we focus on the effect of environmental constraints, such as spatial constraints, on the rate of adaptation to populations of microbes and bacteriophages. To address these questions, we borrow from statistical physics, population genetics and experimental microbiology integrating experimental insights with theoretical models and predictions.

Diana FuscoUniversity of CambridgeMonday, 2nd September, 14:30

How spatial growth constraints evolution: insights from microbes and phages

We have developed 3D cell culture systems from mouse and human fetal pancreas and their engineered equivalents from human pluripotent stem cells as a mean to understand the principles of organogenesis. We have observed how cells self-organize with time and how the culture medium can control the system end-state. We are interested in how cells differentiate to produce endocrine and exocrine cells and how they organize to form a network of ducts, initially a meshwork that evolves towards a tree.

Anne Grapin-BottonMax Planck Institute of Molecular Cell Biology & GeneticsTuesday 3rd September, 14:45

Organoids towards an understanding of self-organization in organogenesis

Our goal is to obtain a physical understanding of the mechanisms by which cells establish and maintain their precise shape and intracellular organization. We focus on the cytoskeleton, a mechanical network of biopolymers and associated proteins that gives the cell its shape and strength. Motor proteins can move over these biopolymers to deliver cargo to specific subcellular compartments or to reorganize the fibers themselves. Our research currently concentrates on the cytoskeleton of neurons, the brain cells whose complex spatial architecture and polarized organization into axons and dendrites are crucial for proper functioning. Obtaining physical insight in the mechanisms that establish polarity, polarized transport and dendrite maturation is important, as neurodegenerative diseases often correlate with altered morphology and distorted intracellular transport.

Lukas KapiteinUtrecht UniversityMonday 2nd September, 14:00

Navigating the neuronal cytoskeleton

My lab is interested in the mechanical control of physiological processes involving the sensation of mechanical stresses. We primarily work with invertebrate model organisms such as Drosophila and C elegans and guide our experiments with theoretical models and simulations. We use advanced imaging and biophysical measurements to infer how me-chanical properties of molecules, cells and tissues governs neuronal biology.

Michael KriegThe Institute of Photonic SciencesMonday 2nd September, 17:05

Exploring force transmission pathways during touch and proprioception

: The goal of our research is to understand - and to be able to predict - phenotypic variation in individuals. This includes understanding mutations, their effects and interactions but also the exceptions i.e. the stochastic and epigenetic causes of variation amongst genetically identical individuals. To understand how mutations interact within and between molecules and how these interactions change across conditions we have performed deep mutagenesis of exons, genes, protein-interactions, regulatory interactions, simple gene networks, and prion-like domains. Excitingly we have recently shown that quantifying the interactions between mutations in a protein provides sufficient information to determine its structure. This provides a new experimental approach for structure determination.

Ben LehnerCentre for Genomic RegulationMonday 2nd September, 10:30

Solving protein structures and understanding genetic interactions using deep mutagenesis

We are interested in the mechanisms that make early development robust and reproducible in the face of fluctuating or imprecise signals. We will present evidence that changes in cell adhesion and tissue morphology modulate the abil-ity of pluripotent cells to respond to extrinsic cues. We propose that changes in cell adhesion are not simply a passive consequence of differentiation, but rather that they actively feed back into the decision making process.

Sally LowellUniversity of EdinburghTuesday, 3rd September, 11:15

Mis-shapes, Mistakes, Misfits and how to avoid them: how do pluripotent cells make the right decisions?

A major obstacle to studying the physical properties and dynamic architecture of organelles and bacteria is their small lateral dimension, which puts them near the diffraction limit of light. My group has developed microscope automation and beam homogenization to enable the collection of large super-resolution datasets. These advances allowed us for the first time to measure the 3D structure of the bacterial z-ring for hundreds of cells across cell cycle and the 3D, multicolor architecture of the centriole. Currently, we are developing deep learning-based algorithms to control “smart microscopes;” automated to adapt data acquisition for optimal spatial or temporal resolution under the constraints set by living systems.

Suliana ManleyÉcole polytechnique fédérale de LausanneTuesday 3rd September, 13:45

Stochasticity and Control in Mitochondrial Division

I study the evolution of pathogens and the host responses that control them. I’m particularly interested influenza viruses and the interplay between evolution and epidemiology.

Colin RussellUniversity of AmsterdamMonday 2nd September, 15:15

Multiscale evolution of influenza viruses

Patchy colloidal self-assembly in animals. Sophisticated scattering optics in animals.

Alison SweeneyUniversity of PennsylvaniaTuesday 3rd September, 10:15

Phase transitions and photonics in cephalopods

(1) What information is encoded in patterns of protein activity, especially the temporal dynamics and spatial gradients of active signaling proteins? (2) How is this information is “read out” by downstream gene/protein networks? (3) During embryo development, how do various spatiotemporal signaling patterns result in sharp boundaries of cell fates and tissue movements? (4) What new techniques and tools (e.g. light-sensitive proteins; new imaging modalities) can be brought to bear on the above questions?

Jared Toettcher Princeton UniversityTuesday 3rd September, 12:00

Optogenetic rescue of a developmental patterning mutant

I am interested in how cells and organisms behave at the cusp of life and “death”, and to reveal how a cell that may appear “dead” restart active form of life.

Hyun YoukDelft University of TechnologyTuesday 3rd September, 9:30

Cells reshape habitability of temperature by helping each other replicate

We theoretically investigate the physical mechanisms that underlay fundamental mechanical processes of cells and tissues. For instance, we study how cells acquire their shape and internal structure and how forces locally generated by cells influence other cells in the environment to govern global changes in cell organization and tissue morphogenesis.

Assaf ZemelHebrew University JerusalemMonday 2nd September, 16:20

Elastic interactions of cells and their role in cellular self-association

ContributedTalks

Abstracts

Shahaf Armon Weizmann Institute of Science

From Contraction Waves to Active Resistance to Rupture - Tissues as Sheets of Relaxation Oscillators

Tuesday 3rd September, 14:30Recently we reported our discovery of fast contraction dynamics in the epithelium of the ancient animal Trichoplax Adhaerens. This not well-known model system has extreme advantages, being the simplest known living animal, made of mono-layered epithelium, practically in 2D. In this animal tissue, in vivo, we observe and quantify activity patterns like intrinsically compressed steady state, chaotic activity and contraction waves. Inspired by these observations, we present our model of tissues as sheets of relaxation oscillators. The new model highlights the unique properties of tissues as active solids with both discrete and continuous properties. It can explain the dynamical patterns we see, but it also predicts excitation in response to stress and dynamic resistance to rupture. I will present both the experimental observations and the model’s theoretical and numerical results. I will mention its possible implications on “higher” tissues, and finally, I will discuss what the model may teach us about the evolution of multicellularity.

Dirk Benzinger ETH Zurich

Pulsatile transcription factor regulation reduces cell-to-cell variability in gene expression

Monday 2nd September, 10:15As a result of stochastic interactions of molecules and fluctuations of the intra- and extracellular environment, gene expression can exhibit substantial variability between cells. Nevertheless, its precise and dynamic regulation is crucial for most biological processes. Recent studies have shown that the activity of many natural transcription factors (TFs) is modulated in a pulsatile fashion. However, the complexity of signal transduction and gene regulation hampers our ability to analyze how the dynamic activity of regulators affects transcription and cellular heterogeneity. Therefore, we have established a synthetic biology approach that makes use of a fast-acting, light-responsive TF to quantitatively study different aspects of transcriptional regulation. Using this approach, we found that pulsatile TF regulation can reduce cell-to-cell variability in protein expression and that expression mean and variability can be independently tuned by adjusting the frequency of input signals. A combination of experiments and mathematical modeling suggests that pulsatile regulation attenuates the effects of two important sources of expression variability: heterogeneity in TF expression, and stochastic transcriptional bursting. We have further combined the synthetic expression system with live-cell quantification of nascent RNAs which allowed us to directly observe how TF activity affects transcriptional dynamics. Our results demonstrate the merit of using well-defined and easily controllable synthetic systems together with simple mathematical models to gain new insight into fundamental biological processes.

Henry De Belly University College London

Membrane tension regulates FGF driven fate choice in embryonic stem cells

Tuesday 3rd September, 11:45Cell fate transitions frequently accompany changes in cell shape and mechanics. Yet the relevance and mechanism of action of these changes to the regulation of cell fate is poorly understood. To probe the interplay between cell shape, mechanics and fate, we focused on embryonic stem (ES) cells, which generally undergo significant spreading during early differentiation. We found that ES cell spreading is highly correlated with cell state and is regulated by a decrease in plasma membrane tension. We show that preventing this decrease in membrane tension obstructs early differentiation of ES cells. Moreover, we found that if cell membrane tension is not decreased, endocytosis of FGF receptors, which leads to increased ERK activity driving the exit from the ES cell state, is significantly inhibited. Importantly, differentiation defects in ES cells where the membrane tension decrease is inhibited can be rescued by increasing Rab5a-facilitated endocytosis. Thus, we show that a mechanically-triggered increase in endocytosis regulates cell fate choice. Our findings are of fundamental importance not only for understanding the role of mechanics in stem cell function, but also for how cell mechanics regulates biochemical signalling.

Serena Ding Imperial College London

Shared behavioral mechanisms underlie C. elegans aggregation and swarming

Tuesday 3rd September, 10:00In complex biological systems, simple individual-level behavioral rules can give rise to emergent group-level behavior. While collective behavior has been well studied in cells and larger organisms, the mesoscopic scale is less understood, as it is unclear which sensory inputs and physical processes matter a priori. Here, we investigate collective feeding in the roundworm C. elegans at this intermediate scale, using quantitative phenotyping and agent-based modeling to identify behavioral rules underlying both aggregation and swarming - a dynamic phenotype only observed at longer timescales. Using fluorescence multi-worm tracking, we quantify aggregation in terms of individual dynamics and population-level statistics. Then we use agent-based simulations and approximate Bayesian inference to identify three key behavioral rules for aggregation: cluster-edge reversals, a density-dependent switch between crawling speeds, and taxis towards neighboring worms. Our simulations suggest that swarming is simply driven by local food depletion but otherwise employs the same behavioral mechanisms as the initial aggregation. We further expand our work by examining swarming at very high densities, and using a bioluminescence bacterial system to visualize and quantify food intake in these collective behaviors.

Maximilian Jakobs University of Cambridge

Microtubule polarity in axons is sorted by a molecular gradient of dynactin

Monday 2nd September, 15:00In neuronal axons, almost all microtubules (MTs) are oriented with their growing end (+end) away from the cell body (+end out). Molecular motor proteins rely on this orientation to efficiently move cellular cargo to the far distal regions of the axon. Despite 30 years of research, the mechanism that establishes this unique MT configuration remains unknown. We here analysed MT growth behavior in Drosophila melanogaster neurons with a novel machine learning algorithm; KymoButler. With the unmatched precision of this approach we discovered that +end out MTs grow for longer times than -end out MTs. Together with an analytical model of microtubule growth, this observation predicted dramatic differences in average MT length, leaving -end out MTs are short and unstable. Additionally, we found evidence that dynactin is responsible for the difference in growth times by promoting growth at the periphery of the cell through a molecular gradient. These findings suggested a simple mechanism that organises axonal MTs. First, +end out MTs are stabilized by distally located dynactin. Subsequently, the short -end out MTs are transported out of the axon, depolymerize, or reorient, leaving only longer +end out MTs in the axon. Our results pave the way towards a deeper understanding of how the cytoskeleton in neurons orients to support molecular transport along the axon, potentially shedding new light on pathologies that are characterised by axonal transport deficiencies such as Alzheimer’s disease.

Nicoletta Petridou Institute of Science and Technology, Austria

Tissue morphogenesis at criticality

Monday 2nd September, 16:50The fitness of embryonic development requires the embryo to find the optimal balance between robustness and adaptability. Such balance is achieved in nature when a system is poised to criticality, meaning that with small changes in its parameters, a transition between the different phase states can occur. Order-disorder phase transitions have been described in macromolecules and gene expression networks, however if they occur in living tissues and what is their role in development is unknown. We have previously described that the onset of morphogenesis in zebrafish relies on an abrupt blastoderm fluidization upon a progressive loss of cell-cell adhesion. By applying tools from statistical physics, we unravel that the blastoderm resides close to a critical value of cell connectivity. Specifically, we map the connectivity of the blastoderm and identify the size of the largest rigid cluster (LRC) through rigidity percolation. Interestingly, at the time point of fluidization we detect a sharp drop in the size of the LRC that takes place upon small changes in cell connectivity, with the latter reaching a critical value where a phase transition occurs. By performing rigidity percolation analysis in different cell types and regions of the embryo we found that the mesodermal germ layer displays a remarkably ordered structure and resides further away from the transition point, ensuring morphogenetic robustness. Intriguingly, altering the non-canonical Wnt signaling pathway, brings the mesodermal tissue to criticality, where the order-disorder transition occurs, compromising its morphogenesis. Overall, we highlight how criticality ensures the fitness of tissue morphogenesis.

Rasha Rezk University of Cambridge

The mechanical heterogeneity in human glioblastoma

Tuesday 3rd September, 14:15Glioblastoma (GBM) is a highly aggressive brain tumour which is currently untreatable due to the genetically intrinsic and extrinsic heterogeneity of this tumour together with its rapid and infiltrative growth. Current treatment strategies have almost exclusively focused on receptor-mediated chemical signalling cascades. However, while any motion, including cell migration and metastasis, is driven by forces, our current understanding of physical interactions between GBM cells and their environment is still very limited. Here we investigated mechanical properties of cells from different tumour regions biopsied using MRI stealth imaging during fluorescence-guided glioma surgery. Using a novel microfluidics device, adhesive forces of live cells were quantified, and cell migration and morphology were obtained using time lapse microscopy. We found that patient-derived primary GBM cells differ in adhesion strength, morphology and cell migration between and within patients’ tumours. The mechanical intra-tumour and inter-tumour heterogeneity is consistent with the heterogeneous genotypic and phenotypic profile characteristic of GBM. Lines from tumour margins were less adherent than lines from tumour cores, suggesting possible mechanisms for the spatial properties of GBM invasion and drug resistance. The variation of GBM cell-matrix adhesion correlated with cell size and cell migration. Marginal cell lines were smaller, adhered less well, and migrated quicker than core cells. This intra-inter tumour heterogeneity could explain recently observed differential responses of patients to adhesion-blocking drugs such as cilengitide, and it strongly suggests that different parts of the same tumours should be treated differently.

Andrew Stannard King’s College London

Mechanically modulating the nuclear translocation of proteins

Monday 2nd September, 10:00Single-molecule force spectroscopy (SMFS) techniques, such as atomic force microscopy, have been widely used to study the mechanical properties of single protein domains by measuring the tensile forces required to unfold them. Using this approach, the mechanical properties of several domains of titin have been studied [1]. In particular, Ig27 (the 27th immunoglobulin-like domain) has become a paradigm and is used as a benchmark of mechanical stability. Here we examine the applicability of these in vitro measurements to an in vivo system - can SMFS aid our understanding of physiological phenomena involving tensile forces? Our experiments investigate whether the nuclear translocation of proteins (passage of cytoplasmic proteins into the nucleus) through the nuclear pore complex (NPC) is regulated by the mechanical stability of the translocating protein. We focus on the translocation of myocardin-related transcription factor A (MRTFA), which localises cytoplasmically in serum-starved cells, but readily translocates to the nucleus upon serum stimulation [2]. With MRTFA fluorescently tagged, its nuclear translocation can be initiated, monitored, quantified, and modelled to extract translocation rate constants. Using this model system, we systematically modify the mechanical stability of fluorescently-tagged MRTFA constructs via the inclusion of domains with varying mechanical stabilities that are well characterised via SMFS. In particular, we concatenate MRTFA-GFP with the wild type and point mutations of Ig27, exhibiting a spectrum of mechanical stabilities in vitro. We find that the rate constant associated with nuclear import displays a clear inverse correlation with the mechanical stability of the translocating construct, demonstrating the mechanoselectivity of the NPC [3]. [1] H. Li et al., Nature 418, 998 (2002) [2] C. Ho et al., Nature 497, 507 (2013) [3] A. Stannard*, E. Infante* et al., Nat. Phys. (in press)

PosterSession

Monday, 2nd September, 17:35

Abstracts

1 Characterisation of the mechano-chemical relationship between actin and micro-tubule cytoskeletons and the active motion of cytoplasmic componentsLaila al-Khatib, Isabel Palacios, Maik Dreschler

Laila al-Khatib Queen Mary University of London

The cytoskeletal network functions as a force-generating entity that is crucial for the proper transport and movement of cellular content. Using the Drosophila oocyte as a model, we aim to understand the relationship between these forces and the motion of cytoplasmic contents. We have shown that movement of vesicles, detected by DIC, is a result of a combination of passive diffusion, active-direct transport and advection (motion by cytoplasmic flows). Advection is attributed to microtubules while a network of cytoplasmic actin filaments, the actin mesh, drives active diffusion. Chemical or genetic elimination of either cytoskeleton disrupts the corresponding processes of vesicle movement.Dynamic processes within the oocyte rely on polarized microtubule arrays. We have now shown that kinesin-mediated cargo transport creates a viscous drag that induces bulk motion of the cytoplasm and lateral displacement forces on microtubules affecting their orientation. Thus, cytoplasmic flows need to be maintained at lower speed and biased random pattern for correct polarisation of the microtubule network. Disrupting the actin mesh leads to increased persistence and speed of flows causing strong defects in orientation. This complex interplay has an effect on the motion of vesicles and organelles within the oocyte, whereby absence of microtubules leads to an absence of flows preventing motion by advection and absence of the actin mesh increases movement by flows, diminishing diffusion-dependent motion. Using differential dynamic microscopy, we aim to characterise the movement and interplay of different populations of vesicles with the cytoskeletal network. Our previous studies have identified different velocities and diffusion coefficients of different Rab vesicles at the same stage of oogenesis, thus we will analyse the link between size and type of motion. We have also included characterisation of mitochondrial motion, manipulating size through the use of fission/fusion mutants.

2 Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regenerationLuigi Aloia, Mikel Alexander McKie, Grégoire Vernaz, Lucia Cordero-Espinoza, Niya Aleksieva, Jelle van den Ameele, Andrea H. Brand, Magdalena Zernicka-Goetz, Stuart J. Forbes, Eric A. Miska and Meritxell Huch

Luigi Aloia University of Cambridge, Gurdon Institute

Upon severe or chronic liver injury, adult cholangiocytes contribute to regeneration by restoring both liver epithelial cell types, hepatocytes and cholangiocytes (or ductal cells). Recently, self-renewing organoid cultures have been established enabling long-term clonal expansion of mature cholangiocytes isolated from primary adult liver tissue. However, the molecular mechanisms by which mature cholangiocytes initiate organoid cultures and regenerate the tissue upon damage remain unknown. Here we describe that adult cholangiocytes undergo epigenetic remodelling mediated, at least in part, by TET1, during formation of liver organoids and upon tissue damage in vivo. TET1 is a member of the TET1/2/3 family of methylcytosine dioxygenases oxidizing the repressive DNA mark 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which has been associated to gene activation. In vitro, TET1 promotes cholangiocyte de-differentiation to a progenitor state by regulating proliferation, stem-cell and regenerative genes. In vivo, TET1 is required for cholangiocyte expansion and cholangiocyte-mediated hepatocyte regeneration upon liver injury. Moreover, decreased TET1 levels in vivo induce liver fibrosis upon chronic damage. Altogether, we demonstrate that adult mature cholangiocytes undergo transcriptional and epigenetic changes enabling them to initiate self-renewing organoid cultures and regenerate the liver after tissue damage induced by a toxic diet.

3 Migration potential of embryonic stem cells at the exit of naïve pluripotencyIrene M. Aspalter, Ewa K. Paluch

Irene Aspalter MRC-Laboratory for Molecular Cell Biology, University College London

Embryogenesis - the formation of a complex highly structured organism - depends on three key events: cell division, cell differentiation, and cell migration. It is unknown when during mammalian development cell migration is initiated. I am investigating the migration potential of murine embryonic stem cells (mESC) at the exit of naïve pluripotency, using microfabricated ex vivo microenvironments, state of the art stem cell biology in combination with live cell imaging naïve mESCs are comparable to epiblast cells in vivo at embryonic day 4.5. Changing the culture conditions in vitro causes naïve mESCs to exit naïve pluripotency and begin the process of differentiation. My data shows that naïve mESCs are able to migrate in an amoeboid fashion in confinement, which is dependent on Myosin IIA. Without confinement naïve mESCs do not migrate. Furthermore, my data shows that the exit of naïve pluripotency is associated with a dramatic shape change from round (amoeboid), to spread (mesenchymal) cells and the formation of focal adhesions. Interestingly, 24h exiting cells are able to migrate in a mesenchymal fashion in a dish without confinement, a process dependent on Arp2/3. This suggests that mESCs undergo and amoeboid-to-mesenchymal transition (AMT) as they exit naïve pluripotency, accompanied by a change in migration mode from specific adhesion independent amoeboid migration to focal-adhesion dependent mesenchymal migration.

4 The contribution of the extra-cellular matrix to tissue mechanics and morphology during developmentRicardo Barrientos, Ewa Paluch, Guillaume Charras, Yanlan Mao

Ricardo Barrientos MRC Laboratory for Molecular Cell Biology, University College London

Extra-cellular matrices are ubiquitous in biological systems, implicated in both mechanics and signalling. The effect of extra-cellular matrix on cell shape in vitro is well established, however much less is known about how the extra-cellular matrix affects tissue morphology in vivo. During development, the extra-cellular matrix surrounding growing tissues must adapt to the changing size and shape of the cellular mass. Physically, whether the extra-cellular matrix contributes to tissue shape depends on the relative mechanical properties between the cells and their extra-cellular matrix. As cells grow, they apply a tension to their surrounding extra-cellular matrix; how the extra-cellular matrix responds depends on their comparative stiffness. When the extra-cellular matrix is stiff compared to cells, stress can be generated at their interface. It is expected that the tissue would have to change shape to minimise this stress. However, any stress build-up may be relaxed over time by remodelling of the extra-cellular matrix. Biologically, stress may induce signals in the cells, allowing dynamic regulation of cell and extra-cellular matrix mechanical properties. This work is concerned with determining how the extra-cellular matrix contributes to the morphology of a growing tissue, using the wing disc of Drosophila Melanogaster as a model system. The wing disc is a larval-stage, highly proliferative, single-cell thick, epithelial tissue folded into a sac, surrounded by a thin extra-cellular matrix. The mechanical properties of this tissue are inferred using a combination of biophysical and genetic techniques. Relative stiffness of cells and their extra-cellular matrix is experimentally measured using atomic force microscopy. Remodelling rates of the extra-cellular matrix are inferred by measuring turnover rates of its components, either in vitro using fluorescence-recovery, or in vivo using temporal genetic knockdown. By combining these measurements with theoretical models of tissues, the relative importance of stiffness and remodelling to tissue shape can be determined.

5 Noisy HES dynamics shaping neurogenesisVeronica Biga, Ximena Soto, Cerys Manning, Jochen Kursawe and Nancy Papalopulu

Veronica Biga University of Manchester

We investigate gene expression dynamics using live tissue containing endogenous HES (Hairy and enhancer of split transcription factors) knock-in reporters across multiple developmental systems and our aim is to understand how noisy gene expression is regulated during the transition to neural differentiation. In order to interpret developmental signals in a dynamical way, we use a combination of live imaging, signal processing and mathematical modelling techniques. In mouse spinal cord, over the course of neural development, cells undergo a transition from an initial “noisy” aperiodic state into an oscillatory dynamic through a process of stochastic resonance (Manning et al. 2019). Remarkably, cells were found to be more frequently oscillatory during downregulation of HES5 and this enables their subsequent differentiation into interneurons rather than motor-neurons. To experimentally manipulate dynamics in vivo we used a Her6 Zebrafish knock-in (HES1 ortholog) (Soto, Biga et al.) and showed that cells transition towards reduced noise (quantified as high lengthscale and Fano factor) as they differentiate. However, in the absence of known repression by miR-9, noise is amplified in Her6, leading to a reduction in number of oscillators through an inverse stochastic resonance effect. The lack of miR-9 regulation and presence of higher noise levels is associated with impaired progression towards neural differentiation and mis-expression of target genes. Our findings shed light onto hidden dynamics of noise control at single cell level during neurogenesis and highlight the importance of time-resolved quantitative techniques. Related papers: Manning et al. (2019) Quantitative single-cell live imaging links HES5 dynamics with cell-state and fate in murine neurogenesis. Nature Communications Vol 10(1): 2835; Soto, Biga et al. miR-9 mediated noise optimization of the her6 oscillator is needed for cell state progression in the Zebrafish hindbrain. bioRxiv https://www.biorxiv.org/content/10.1101/608604v1

6 Identifying how intestinal cell fate is controlled by Integrin-mediated mechano-transductionJerome Bohere, Golnar Kolahgar

Jerome Bohere University of Cambridge

To maintain gut size and function, intestinal stem cells (ISC) integrate dynamic chemical and mechanical signals from the surrounding cells and environment to decide whether to proliferate or differentiate. Failure to do so may result in loss of tissue integrity and development of degenerative diseases. Our aim is to characterize in vivo the mechanisms causing mechanical signals to regulate ISC proliferation. We take advantage of the structural and genetic simplicity of the Drosophila gut to investigate how integrin-mediated mechano-transduction regulates stem cell fate as the whole gut remodels in response to physiological physical constraints. Integrins are mechano-sensitive transmembrane proteins linking the extracellular matrix to the cytoskeleton via associated proteins such as Vinculin, ultimately regulating gene expression and thus cell behavior. Integrin subunits are enriched in many mammalian adult stem cells, but functional characterization remains complicated due to genetic redundancy. In Drosophila, genetic ablation of the ubiquitous βPS Integrin subunit in gut stem cells prevents proliferation, however the downstream molecular mechanisms are unknown and the mechano-sensitive role of integrins remain to be addressed. By combining genetic tools, clonal analyses and measurements of tissue stiffness, we show that homeostatic ISC fate relies on distinct requirements of the adhesive and signaling components of the Integrin complex. We are now testing if similar mechanisms regulate stem cell proliferation in regeneration and tumour growth.

7 A mechanochemical model to explain patterning transitions in the cell cortexDaniel Boocock, Edouard Hannezo

Daniel Boocock IST Austria

There is a long-standing interest in understanding the mechanisms behind pattern formation in biological systems with an increasing realisation that many important phenomena involve complex interplays between mechanics and chemistry. At the scale of the cell cytoskeleton a wide variety of dynamic patterning behaviours are seen ranging from homogeneous oscillations to chaotic motion and merging of foci. Mechanical instabilities have been proposed to drive this patterning behaviour although it was recently shown that the actomyosin regulatory circuit is an intrinsic chemical oscillator exhibiting oscillations even when decoupled from mechanics [Qin et al., 2018]. This raises the question of how mechanics and biochemistry interact. To explore this question we formulate a mechanochemical description of the cortex involving a simple chemical oscillator and find that it reproduces the different patterning behaviours seen in the real system. The precise behaviour is determined by just two parameters: one related to the rate of chemical turnover and a second to the strength of myosin contractility. This neat separation gives us insight into the different effects of chemistry and mechanics and allows us to draw a simple phase diagram for the different behaviours. We use these results to argue that the breaking of oscillations coincides with the point at which actomyosin contraction is strong enough to induce net fluxes inside the cortex. We apply this to experimental findings on drosophila to determine where in phase space different living systems reside. References: X. Qin, E. Hannezo, T. Mangeat, C. Liu, P. Majumder, J. Liu, V. Choesmel-Cadamuro, J. A. McDonald, Y. Liu, B. Yi, et al. A biochemical network controlling basal myosin oscillation. Nature communications, 9(1):1210, 2018.

8 Energy flux and the maintenance of polarity by the PAR networkJoana Borrego-Pinto, Nate Goehring

Joana Borrego-Pinto The Francis Crick Institute

The PAR proteins are conserved proteins that specify the polarity axis of diverse animal cells such as neurons, epithelia, migrating cells, oocytes and embryos, such as the C. elegans zygote. Here, PAR proteins partition into two distinct plasma membrane domains, anterior and posterior, which in turn polarises the cytoplasm, and consequently ensures the unequal segregation of cell fate determinants at the first asymmetric cell division. These domains can be maintained autonomously by mutual exclusion between opposing PARs, which heavily relies on phosphorylation and therefore energy. As most animals obtain their energy via aerobic respiration, alterations on oxygen levels could easily impact the energy available for cellular processes. Interestingly, during anoxia (oxygen absence), several species enter in a state of suspended animation. This state is reversible when normal conditions of oxygen return, although long anoxic periods will negatively impact survival rates. We are studying the consequences of energy depletion in polarity in anoxic conditions, using the C. elegans zygote as model organism. Given the high energy requirements for maintaining polarity in this system, this would suggest that polarity would collapse during anoxia. Our data show posterior PARs lose asymmetry, as expected. In contrast, anterior PARs maintain a residual asymmetry. Upon return of normal conditions of oxygen, both anterior and posterior PARs reform the previously established domains and embryonic development proceeds as normal. We are currently exploring how this asymmetry is maintained during anoxia and whether this population could provide a spatial memory of the polarized state, which allows the embryo to quickly recover its homeostasis.

9 Phase-space geometry of mass-conserving reaction-diffusion systemsFridtjof Brauns, Jacob Halatek, Erwin Frey

Fridtjof Brauns Arnold Sommerfeld Center for Theoretical Physics, LMU, Munich

Experimental studies of protein pattern formation (both in vivo and in vitro) have stimulated new interest in the dynamics of reaction--diffusion systems. However, a comprehensive theoretical understanding of the dynamics of such highly nonlinear, spatially extended systems is still missing. Here we show how a description in phase space, which has proven invaluable in shaping our intuition about the dynamics of nonlinear ordinary differential equations, can be generalized to mass-conserving reaction--diffusion (McRD) systems. We present a comprehensive theory for two-component McRD systems, which serve as paradigmatic minimal systems that encapsulate the core principles and concepts of our framework. The key insight is that shifting local (reactive)---controlled by the local total density---give rise to concentration gradients that drive diffusive redistribution of total density. We show how this dynamic interplay can be embedded in the phase plane of the reaction kinetics in terms of simple geometric objects: the reactive nullcline (line of reactive equilibria) and the diffusive flux-balance subspace. On this phase-space level, physical insight can be gained from geometric criteria and graphical constructions---the effects of nonlinearities on the global dynamics are simply encoded in the curved shape of the reactive nullcline. In particular, we show that the pattern-forming `Turing instability’ in McRD systems is a mass-redistribution instability, and that the features and bifurcations of patterns can be graphically determined by a flux-balance construction on the reactive nullcline. In the outlook we detail several concrete approaches to generalize the local-equilibria framework to systems with more components, exhibiting more complex phenomena, and beyond strictly mass-conserving dynamics.

10 Robustness of microtubule self-organisationAleksandra Z. Plochocka, Alexander M. Davie, Natalia A. Bulgakova, Lyubov Chumakova

Natalia Bulgakova University of Sheffield

The microtubule cytoskeleton is a dynamic network of “highways” along which cellular components, e.g. proteins, are transported to their biologically relevant locations. Its organization is essential for correct cellular and organism function, whereas perturbing it results in pathologies. Microtubules, which build this network, are inherently dynamic polymers which undergo phases of growth (polymerisation) and shrinkage (depolymerisation). Numerous studies show that dynamics of individual microtubules are affected by environment, e.g. temperature, and genetics, which vary between organisms. An outstanding question is how, given a broad range of possible dynamic behaviours, do cellular components reliably reach their target locations in correct amounts? Here we analyse cytoskeleton self-organization in epithelia, an omnipresent tissue in eukaryotes, and study it via mathematical modelling and genetic manipulations of Drosophila embryo. We demonstrate that the microtubule network self-organization depends on cell geometry alone and is robust on the tissue scale. In particular, the large-scale stochastic simulations suggest that the self-organization is robust for a wide parameter range, while genetic manipulations of Drosophila embryo epithelial cells confirm the robustness in vivo. Finally, we develop a minimal mathematical model that suggests that the origin of robustness is the generic separation of scales in microtubule dynamics. These results suggest that simple mathematical principles underlie the robustness of self-organization of this complex biological system, providing an elegant mechanism for ensuring correct function and maintenance of epithelia despite their varying geometries and environments.

11 Net Flux and Chill - Modelling Collagen and the ECMBen Calverley, Adam Pickard, Yinhui Lu, David Holmes, Oliver Jensen, Tom Shearer, Karl Kadler

Ben Calverley Wellcome Centre for Cell Matrix Research, University of Manchester

In tissues like tendon, collagen forms packed bundles or fascicles of approximately cylindrical fibrils. It has been shown that mouse tendon fibril size distribution is diurnally rhythmic. What mechanical effects and/or properties does this geometry bring to the tendon? We use micro-scale cryo-electron microscopy imaging data to analyse and predict macroscopic tendon functions such as viscoelasticity.

12 Deep Learning Drug-Treated Phenotypes from High-Throughput ImagingHenry Cavanagh, Robert Endres, Rob Lind, Andreas Mosbach, Gabriel Scalliet

Henry Cavanagh Imperial College London

Microorganisms’ phenotypes divulge rich information regarding their surroundings, health states and adaptation strategies; tractable and interpretable representations of these are therefore highly desirable. The recent advances in deep learning and high-throughput imaging enable key shape variations to be captured in low dimensional, feature-based representations. We aim to use such representations to develop a deep learning, biological physics hybrid model that provides mechanistic insights into the effects of experimental drugs on microorganisms. Images of in vitro fungicide-treated spores of Phakopsora pachyrhizi, a devastating fungus that can reduce yields of the economically vital soybean crop by 80%, have been provided by Syngenta. To date, we have been using variational autoencoders to acquire disentangled phenotype representations from the images, and we aim to subsequently extend the model to 3D cell inputs. The proposed technology has the potential to impact drug discovery in the medical and agricultural sectors, as well as fundamental biological research into sensing and signalling.

13 Evolution of complex systems: thermodynamics of switching in multistable systemsJacob Cook, Robert G. Endres

Jacob Cook Imperial College London

Multistable nonequilibrium systems are abundant outcomes of nonlinear dynamics with feedback but still relatively little is known about what determines the stability of the steady states and their switching rates in terms of entropy and entropy production [1]. Here, we will link fluctuation theorems for the entropy production along trajectories with the action obtainable from the Freidlin–Wentzell theorem to elucidate the thermodynamics of switching between states in the large volume limit of multistable systems. We find that the entropy production at steady state plays no role, but the entropy production during switching is key. Additional stabilising and destabilising effects arise from the steady-state entropy and diffusive noise, respectively. In addition to ecosystems, this framework may be applicable to evolution via an analogy between entropy production and fitness flux [2]. Our framework then suggests that states in a fitness landscape requiring greater cumulative fitness flux to reach should be expected to be more dynamically stable.[1] R.G. Endres, Sci. Rep. 7, 14437 (2017). [2] V. Mustonen and M. Lässig, PNAS 107, 4248 (2010).

14 Bridging time scales through transfer operators: variability and control in C. elegans locomotionAntonio Carlos Costa, Greg Stephens

Antonio Carlos Costa Vrije Universiteit Amsterdam

Animal behavior exhibits multiple time scales: from the fine scale movements dictated by the coordinated activity of neurons, to longer time scale processes that modulate behavior like starvation or mating. Can we extract these hidden processes from the movement dynamics alone? In this work, we details a novel approach that explores the spectral properties of the transfer operator built from the postural dynamics to extract a hierarchy of behavioural modes that evolve over multiple time scales, as well as coherent sets that naturally correspond to interpretable behaviors. This effective multi-scale model of the dynamics provides a framework for the study of variability and unpredictability of movement as well as a means to understand control mechanisms over multiple scales.

15 Membrane shape instability and pattern formation of surface bound proteinsGeorge Dadunashvili, Floor de Jong, Storm van de Linde, Afshin Vahid, Timon Idema

George Dadunashvili Delft University of Technology

Membranes in biological systems exhibit a huge variety of shapes which are related to their function. Much of this function is performed by surface-associated proteins, which themselves influence membrane shapes. Understanding this reciprocal relationship is thus of great importance for understanding biological function at the cellular level. Our group has already investigated this relationship from several angles, e.g. in a recent publication [1], we show, in collaboration with experimental groups, that crescent shaped proteins can both act as curvature sensing and as curvature inducing, depending on their concentration. In this work we look at the limit of very large number of curvature inducing proteins. To do so, we replace the notion of individual particles on the membrane with a concentration field coupled to its spontaneous curvature. A similar approach has already been used to successfully describe traveling wave propagation in oocytes [2]. We start by investigating under which conditions a cylindrical membrane tube covered with proteins becomes unstable. After finding the set of conditions for instability we utilise finite difference and finite element methods to analyse the instabilities in the tube shape and protein distributions. We find that curvature plays a significant role in stability of the system and proteins start demixing on a tube while they would not in a flat geometry. References: [1] Helle, S. C. J., Feng, Q., Aebersold, M. J., Hirt, L., Gruter, R. R., Vahid, A., Sirianni, A., Mostowy, S., Snedeker, J. G., Andela, S., Idema, T., Zambelli, T., and Kornmann, B. Mechanical force induces mitochondrial fission. eLife 6 (2017), e30292. [2] Miller, P. W., Stoop, N., and Dunkel, J. Geometry of Wave Propagation on Active Deformable Surfaces. Physical Review Letters 120, 26 (2018), 268001.

16 Reverse engineering cell competition using time-lapse microscopy and deep learning. Nathan Day, Alan Lowe, Guillaume Charras

Nathan Day University College London

Cell competition is broadly defined as a quality control mechanism that results in less-fit “loser”cells being eliminated from a biological tissue. This mechanism impacts a wide variety of different physiological and pathological scenarios, from senescence to tumorigenesis. Previous studies into cell competition have typically characterised it in regard to population-level dynamics, but this approach neglects the individualised role of the cell as an independent agent in this process. How are the rules of cell competition going to be understood if we don’t observe the actions of individual players? In this project, time-lapse microscopy and deep learning image analysis allows for the labelling of individual cells and classification of their phase in the cell life cycle. Bayesian tracking then maps the path the cell takes throughout its lifetime. Studying heterogeneous monolayers of epithelial cells, this approach yields data that allows for an investigation into the behaviour of a tissue to a high level of temporal and spatial resolution; all the cells imaged are followed and annotated at every timestep throughout their entire lifetime. Coupling these tools with novel imaging techniques, such as quantitative phase microscopy and 3D single plane illumination microscopy, both with fluorescent labelling capabilities, allows for a deep level of insight into the morphological and biochemical properties of each cell during every scenario of competition. The resulting impact of this study will be to improve our understanding of the mechanisms of cell competition through the highly-resolved data sets acquired. The dependencies of cellular density and different biochemical marker expression on the local cellular configuration will be thoroughly investigated. Using this big data, the overarching aim of the project is to reverse engineer cell competition purely from behavioural observation, eventually building a neural network-based predictive model for cellular fate.

17 Is biological growth driven by stress or strain?Alexander Erlich, Gareth W. Jones, Françoise Tisseur, Derek E. Moulton, Alain Goriely

Alexander Erlich Université Grenoble Alpes

In many biological systems, cell, tissue, and organ growth are influenced by mechanical cues. Locally, cell growth leads to a mechanically heterogeneous environment as cells pull and push their neighbors in a cell network. Despite this local heterogeneity, at the tissue level, the cell network is remarkably robust, as it is not easily perturbed by changes in the mechanical environment or the network connectivity. Through a network model, we relate global tissue structure (i.e. the cell network topology) and local growth mechanisms (growth laws) to the overall tissue response. Within this framework, we investigate the two main mechanical growth laws that have been proposed: stress-driven or strain-driven growth. We show that in order to create a robust and stable tissue environment, networks with predominantly series connections are naturally driven by stress-driven growth, whereas networks with predominantly parallel connections are associated with strain-driven growth.

18 Enhancer priming enables fast and sustained transcriptional responses to Notch signalingJulia Falo-Sanjuan, Nicholas C Lammers, Hernan G Garcia, Sarah Bray

Julia Falo-Sanjuan University of Cambridge

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

19 Towards high-throughput DNA synthesis in a silicon-based MEMS “virtual thermal well”-arrayAndrew Ferguson, Gary Skinner, Vijay Narayan , Yen-Chun Lin, Blair Kirkpatrick, Matthew Hayes, Albert Prak

Andrew Ferguson Evonetix Ltd

Evonetix is a Cambridge-based start-up company working on a revolutionary new technology to synthesise high-fidelity DNA at scale. Our technology is based on a silicon MEMS chip with a large number of reaction sites that facilitate multiple parallel synthesis channels. We operate a modified phosphoramidite cycle on these sites and combine with a proprietary error detection scheme to enhance yield. Our silicon MEMS chip consists of an array of heaters that operate in a common fluidic environment to create distinct and independent thermal wells – fluid volumes centred around the heaters whose temperature can be controlled precisely. Despite not being separated by physical barriers, the wells can be addressed and tuned independently, even under conditions of flowing liquid. Chemical reactions can be reversibly enabled/disabled on each site using temperature control. Based on our physical simulations, we have manufactured prototype MEMS chips whose performance compared very favourably to predictions. The experimental tests included fluorescent microscopy to measure the volumetric and surface temperature profiles within a thermal well. For the former we used rhodamine B, a dye with temperature-sensitive fluorescence, suspended in the fluid and confocal microscopy to map the temperature within the well. For the latter we used the dye Cy5 covalently tethered to the chip surface, enabling the thermal environment at the surface to be probed. In our poster we will outline the development of our MEMS chip, show its characterisation by means of fluorescent microscopy and discuss its application to the synthesis of DNA.

20 Ectopic expression of Lamin A in murine embryonic stem cells blocks neural lineage specificationGeorge Wylde, Mehdi S Hamouda, Celine Labouesse, Kevin J. Chalut

Mehdi Hamouda Wellcome-MRC Cambridge Stem Cell Institute

Embryonic Stem (ES) cells are defined by their competence to contribute to every germ layer. Differentiation and lineage commitment are encoded into gene regulatory networks and determined by a precise sequence of signalling events and epigenetic changes. Activation of lineage markers is also dependent on their nuclear localisation and tethering to the nuclear lamina1,2. Notably, ES cells do not express A-type lamins, which contribute to nuclear stiffness and to chromatin tethering. To better understand the role of the nuclear envelope in gene regulation during differentiation, we ectopically expressed Lamin A (LmnA) in ES cells, and used qPCR, ChIP and staining methods to look at expression, histone methylation and locus localisation of early neural lineage marker Sox1. We found that LmnA overexpression during a critical time window of early differentiation (first 20hrs) impairs downstream neural lineage commitment at a later stage (days 3-4). We also observed an enrichment of repressive histone mark H3K27me3 at the promoter of Sox1, suggesting some interaction between the nuclear lamina and the Polycomb machinery. Finally, 3D DNA-FISH assays showed that LmnA overexpression results in mislocalisation of Sox1 locus closer to the nuclear periphery. Together, these results suggest that ectopic expression of LmnA hinders the chromatin reorganisation of early differentiation3, resulting in defects in lineage commitment. To test whether this is only due to chromatin tethering or also to changes in nuclear mechanics, we are using perturbations of nuclear envelope proteins to abolish force transmission to the nucleus in differentiating ES cells. References: 1.Solovei, I., Wang, A. S., Thanisch, K., Schmidt, C. S., Krebs, S., Zwerger, M., ... & Herrmann, H. (2013). LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell, 152(3), 584-598. 2. Peric-Hupkes, D., & van Steensel, B. (2010, January). Role of the nuclear lamina in genome organization and gene expression. In Cold Spring Harbor symposia on quantitative biology (Vol. 75, pp. 517-524). Cold Spring Harbor Laboratory Press. 3. Marks, H., Kalkan, T., Menafra, R., Denissov, S., Jones, K., Hofemeister, H., ... & Stunnenberg, H. G. (2012). The transcriptional and epigenomic foundations of ground state pluripotency. Cell, 149(3), 590-604

21 A time delay model of “micro-patterning” arising from HES5 dynamicsVeronica Biga, Jochen Korsawe, Nancy Papalopulu, Paul Glendinning

Joshua Hawley University of Manchester

During development of the central nervous system, neural stem cells establish a network of cell-to-cell communication through Notch-Delta receptor-ligand interactions which is important for ensuring correct spatiotemporal control of proliferation of the neural stem cell population (Shimojo et al., 2011 Front Neurosc). Notch Delta signalling acts as a lateral inhibition process, whereby a cell that highly expresses Delta ligand will repress Delta production in neighbouring cells and classically this results in a steady state “checkerboard-like” pattern (Collier et al., 1996 J Theor Biol). We focus on spinal cord tissue, where we identified that the Notch target gene HES5 (Hairy and enhancer of split-5), a transcription factor responsible for preventing premature neural differentiation, can exhibit periodic activity of 3-4h (Manning et al. 2019 Nat Comms) due to its auto-repressive nature. The presence of oscillatory expression downstream of Notch complicates our understanding of cell-to-cell interactions and how dynamic signalling relates to cell fate decisions at the single cell level with respect to the decisions of neighbouring cells. To understand these complex interactions, we generate a reduced model resulting in a system of effectively coupled autonomous oscillators, integrating stochasticity and time delays at the single cell level, along with intercellular time delays and lateral inhibition at a multicellular level. This builds upon existing models (Manning et al. 2019; Phillips et al. 2016 eLife) to explain the emergence of previously unreported local correlations (“micro-patterning”) between neighbouring cells. We find under certain parameter values pertaining to coupling strength and intercellular time delays, that the tissue can give rise to local correlations between cells as repeated bands of high HES5 expression (3-5 cell widths) that intersperse with bands of low HES5 expression. To understand if these dynamics resemble the spinal cord live data we use a combination of frequency analysis and statistical techniques.

22 Interkinetic nuclear migration - a stochastic process constrained by tissue architectureAnne Herrmann, Afnan Azizi, Salvador J. R. P. Buse, Yinan Wan, Philipp J. Keller, William A. Harris, and Raymond E. Goldstein

Anne Herrmann University of Cambridge

In developing pseudostratified epithelia, nuclei move repeatedly between the apical and basal surfaces of cells. This process is termed interkinetic nuclear migration (IKNM) and has been studied extensively in the brain, retina and spinal cord of multiple organisms. But despite these efforts many questions about the precise mechanism of IKNM remain. Based on in vivo light sheet microscopy we develop a quantitative model for the phenomenological properties of IKNM in the retinal system. Both the data and our model support the hypothesis of IKNM being a stochastic process during the majority of the cell cycle. Furthermore, our model reveals the remarkable and previously overlooked importance of simple physical constraints imposed by the overall tissue architecture. Because IKNM has been suggested to fulfil a regulatory role for retinal cell differentiation, our results have important implications for understanding proper eye development. Moreover, our findings will inform future work on IKNM in other organs and on the developmental regulation in these systems.

23 RHEOS - making the analysis of rheological data simpler, faster and more reproducibleBonfanti, A, Kaplan JL, Kabla, A

Alessandra Bonfanti University of Cambridge

Mechanics is increasingly recognised as a key player in the physiology and regulation of biological processes. Although instruments and techniques exist to quantify mechanical properties, analysing experimental data and extracting materials parameters remains a challenging tasks. RHEOS is an open source project aiming to address that issue by providing rich rheological analysis toolkit to interpret viscoelastic material data, biological or otherwise. RHEOS has a particular emphasis on linear rheological models containing fractional derivatives which have demonstrable utility for the modelling of biological materials but have hitherto remained in relative obscurity – possibly due to their mathematical and computational complexity. The package can be freely downloaded at https://github.com/JuliaRheology/RHEOS.jl

24 The Role of Extracellular Mechanics in Skin Tissue Engineering and AgeingEve Hunter-Featherstone, Gabrielle Saretzki, Charles C Bascom, Martin W Goldberg, Iakowos Karakesisoglou

Eve Hunter-Featherstone Durham University

Biomechanical stimuli play a key role in controlling the gene expression, cytoskeletal organisation and ultimately the fate of mammalian cells. Mechanical cues imparted to a cell by its surroundings are converted into biochemical responses that can alter proliferation, differentiation, and trigger apoptosis. The epidermis of the skin has a high turnover rate, with biomechanical input a key factor in enabling self-renewal and wound healing to ensure the maintenance of the barrier function. Despite this, mechanotransduction is often overlooked in the development of in vitro skin models. We hypothesize that biomechanical conditioning during culture will favourably impact skin tissue assembly in vitro.HaCaTs and primary human keratinocytes were cultured on 50, 8 and 4 kPa biomimetic hydrogels to simulate different niches in the epidermal environment. Cells were observed to proliferate more slowly on softer dishes and presented upregulated differentiation markers and increased cell density. A group of chosen mechanoproteins were observed to decrease in correlation with lower gel stiffness. Cells primed on 50 kPa substrates produced thicker, more organised epidermal equivalents than those grown on tissue culture plastic. Thus, priming cells on biomimetic surfaces could be the key to producing a more in vivo-like 3D skin model.

25 Microtubule polarity in axons is sorted by a molecular gradient of dynactinMaximilian A. H. Jakobs and Kristian Franze

Maximilian Jakobs University of Cambridge

In neuronal axons, almost all microtubules (MTs) are oriented with their growing end (+end) away from the cell body (+end out). Molecular motor proteins rely on this orientation to efficiently move cellular cargo to the far distal regions of the axon. Despite 30 years of research, the mechanism that establishes this unique MT configuration remains unknown. We here analysed MT growth behavior in Drosophila melanogaster neurons with a novel machine learning algorithm; KymoButler. With the unmatched precision of this approach we discovered that +end out MTs grow for longer times than -end out MTs. Together with an analytical model of microtubule growth, this observation predicted dramatic differences in average MT length, leaving -end out MTs are short and unstable. Additionally, we found evidence that dynactin is responsible for the difference in growth times by promoting growth at the periphery of the cell through a molecular gradient. These findings suggested a simple mechanism that organises axonal MTs. First, +end out MTs are stabilized by distally located dynactin. Subsequently, the short -end out MTs are transported out of the axon, depolymerize, or reorient, leaving only longer +end out MTs in the axon. Our results pave the way towards a deeper understanding of how the cytoskeleton in neurons orients to support molecular transport along the axon, potentially shedding new light on pathologies that are characterised by axonal transport deficiencies such as Alzheimer’s disease.

26 Evolutionary Dynamics of Viral Populations with Density-dependent DispersalSherry Jiatong Jiang and Diana Fusco

Sherry Jiang University of Cambridge

In this project, evolution dynamics of bacteriophages interacting with spatially-distributed bacteria is studied via Monte Carlo simulations. The system has been previously described using a travelling wave solution given by a Fisher-Kolmogorov reaction-diffusion equation with density independent diffusion. New experimental evidence shows, however, that the dispersal rate and the growth rate terms may be in fact density-dependent, similar to cooperative populations. Using agent-based modelling, we will be able to characterize this effect in the population and evolutionary dynamics of phages. The decay of genetic diversity will be characterized and compared to other range expansion models that accommodate cooperation. The key parameters that control this effect will also be determined.

27 Regulation of notochord size and shape in mouse developmentKasumi Kishi, Anna Kicheva, Edouard Hannezo

Kasumi Kishi IST Austria

The notochord is a transient rod-like structure that extends along the anterior-posterior body axis and lies underneath the neural tube during embryonic development in vertebrates. The notochord secretes multiple proteins, including the morphogen sonic hedgehog (Shh), and has a well-established role in patterning the neural tube and somites. During body axis extension, the notochord, somites and neural tube extend in a coordinated fashion. However, how this coordination is achieved remains poorly understood. One reason for this is that the cell behaviours that contribute to notochord morphogenesis are still unresolved. In particular, it is unclear how cell division, cell death and active cell migration and cell specification each contribute to notochord extension. Here, we propose to use live imaging of mouse embryo explants to study the morphogenetic behaviours of the notochord. Preliminary experiments in mouse and chick revealed directional movement of cells in the notochord, relative to the surrounding tissues. At E9.5 of mouse development, these directed movements cannot be explained by addition of new cells from the node to the notochord, and we are testing whether this movement arises via active and collective cell migration or the passive displacement of cells as a result of cell divisions. To this end we will collect quantitative data on the cell division and movement patterns in the notochord, and aim to reconcile these measurements with the overall shape changes of the notochord over 48h of development using an active fluid model. Perturbation experiments will further allow us to test how distinct cell behaviours contribute to regulating the notochord length and diameter, and how they affect the surrounding tissues.

28 Influence of the mechanical environment on neuronal maturationEva Kreysing, Helene Gautier, Thora Karadottir, Kristian Franze

Eva Kreysing University of Cambridge

During the development of the nervous system, neurons extend long axons as well as shorter and highly branched dendrites to connect to other cells. Once integrated in the neuronal network, neurons mature and the conductivity of their cell membrane changes for certain ion types, resulting in their electrical activity. While mechanical interactions between neurons and their environment are crucial for axon growth and pathfinding, the influence of mechanical signals on neuronal maturation is currently poorly understood. Here, we cultured primary hippocampal neurons on polyacrylamide gels of different stiffness and studied how substrate mechanics impacts the electrical maturation of the cells using Patch-Clamp measurements. Currents through voltage-gated sodium channels, potassium channels, as well as spontaneous activity all started several days earlier in neurons cultured on soft substrates if compared to stiff substrates. These differences in the onset of electrical activity were accompanied by increased synaptic densities on soft substrates as assessed by immunocytochemistry. Our results suggest that mechanical signals play an important role in neuronal maturation, and that local brain tissue stiffness may thus be a key parameter for proper brain development.

29 Advanced Imaging of amyloid-ß protein aggregationMeng Lu, Neil Williamson, Ajay Mishra, Claire H. Michel, Clemens F. Kaminski, Alan Tunnacliffe, Gabriele S. Kaminski Schierle

Meng Lu University of Cambridge

The misfolding and aggregation of intrinsically disordered proteins is a hallmark of neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease. Although a variety of methods have been applied to study protein aggregation in different conditions, the details of its self-assembly process are largely unknown. A key requirement to understand this is to capture the fast assembly of the aggregation-prone proteins and the heterogeneous structures of the aggregate species at molecular level resolution, notably inside the cell. The rapid development and proliferation of advanced imaging, especially super-resolution imaging, in different areas of cell biology have been revolutionising our observation and understandings of structural organisation, dynamics of intracellular macromolecules and organelles. Here, we constructed stable cell lines expressing aggregation-prone proteins and applied Structural Illumination Microscopy (SIM) to study the kinetics of the protein aggregation and its heterogeneous structural forms in live samples. These studies reveal how proteins aggregate in an unprecedented high spatial-temporal resolution, such as the kinetics of aggregate seeding and expansion, the motions and distribution, and their structural change. Particularly, we identified five development stages of intracellular amyloid ß (Aß) aggregate - oligomers, single fibrils, fibril bundles, clusters and aggresomes - that underline the heterogeneity of these Aß42 aggregates and represent the progression of Aß42 aggregation within the cell. In another study based on fast SIM, we applied single particle tracking to analyse the rapid motion of intracellular aggregates and demonstrated that aggresome is chiefly driven by diffusion of small aggregate clusters. These studies uncover the structural progression of protein aggregates in the cell.

30 Physics of Starving Matter: Investigating the physical properties of polyphosphate granules in starving P. aeruginosa to elucidate their functionSofia Magkiriadou, Lisa Racki, Suliana Manley

Sofia Magkiriadou École polytechnique fédérale de Lausanne

When starved, many bacterial species make spherical granules that contain polyphosphate. In many bacteria, these granules are important for starvation survival [Rao 2009]; and in some, polyphosphate is required for efficient cell cycle exit during starvation [Boutte 2012, Racki 2017]. Despite the ubiquity and importance of these structures, very little is known about either their composition or their function: their small size, on the order of 200 nm at most, makes them very difficult to observe with optical microscopy. In search of further clues, we investigate the physical properties of polyphosphate granules and their growth, using transmission electron microscopy images of Pseudomonas aeruginosa at various time points after the onset of starvation. With this information at hand, we ask: are these granules solid, as their colloquial name might suggest, or can their evolution be described with a model for droplet nucleation and growth? Moreover, polyphosphate granules appear, on average, to be regularly spaced. Can these spatial correlations be explained as the result of random packing of spheres in a confined space? Or are these correlations too strong, implying that the granules might be interacting with one another or with an underlying structure? Elucidating such basic physical facts can give insight into both the chemical composition as well as the biological function of polyphosphate granules in starving bacteria. This knowledge will not only contribute to fundamental understanding in biology, but is also very pertinent to the advancement of novel techniques to combat antibiotic tolerance.

31 The role of multi-tissue mechanics in embryo axis elongationSusannah McLaren, Ben Steventon

Susannah McLaren University of Cambridge

How do progenitor cells find their way into the correct tissues as an embryo is developing? A mass of progenitors exists within the tailbud of the developing Zebrafish embryo. These progenitors move out of the tailbud and contribute to specific tissues as the embryo develops, contributing to the elongation of its body axis. Whilst the biochemical cues contributing to the specification and exit of these cells from the tailbud have been studied, the impact of mechanical forces on the movement of these cells, and in channelling them out of the tailbud, is not well understood. Here, we present data suggesting that the elongation of the notochord, coupled to the notochord progenitors, impacts on the exit of a population of progenitors from the tailbud. By perturbing the elongation of the notochord, we investigate how forces arising from its movement impact on presomitic mesoderm progenitor movements in the tailbud, and on their contribution to the presomitic mesoderm. In doing so we hope to build a clearer picture of how multi-tissue mechanical interactions shape overall embryo morphogenesis.

32 Single cell imaging for characterisation of cell competition in epithelial monolayersJasmine Michalowska, Anna Bove, Daniel Gradeci, Shiladitya Banerjee, Guillaume Charras & Alan LOWE

Jasmine Michalowska University College London

In cell competition the viability of a loser cell is determined by context. Our central hypothesis is that the outcome of cell competition at the single-cell level is governed by the unique neighbourhood of that cell. We do not understand how the topology, composition and time evolution of the neighbourhood defines a cell’s fate. Here we use long-term fluorescence time-lapse microscopy and machine learning to quantitatively compare mechanical and biochemical competition in MDCK epithelial cell monolayers. Using co-cultures of wild-type cells and cells depleted of the polarity protein scribble, or expressing Rasv12, we determine how local density and cell identity affects the outcome of competition experiments. We track the proliferation of winner cells and apoptosis of loser cells in various seeding densities, and analyse on a single-cell scale how the neighbourhood in these mixed populations determines the outcome of competition.

33 A continuous nanoscale tubular membrane network, axon endoplasmic reticulumCahir J O’Kane, Lu Zhao, Juanjo Perez Moreno, Megan Oliva, Belgin Yalçın, Martin Stofanko, Niamh C O’Sullivan, Valentina Baena, Mark Terasaki

Cahir O’Kane University of Cambridge

Endoplasmic reticulum (ER) is an intracellular membrane-bound organelle, ubiquitous and continuous throughout cells. In the axons of neurons, it comprises a network of of long membranous tubules that surround a narrow aqueous lumen. It has physical features that must define its function: continuity over distances that are enormous on a subcellular scale, up to 1 m in humans; and highly curved tubules, with a tiny aqueous lumen. Human disease genetics suggests roles for ER architecture in axon maintenance: mutations in several ER-shaping proteins are causative for the axon degeneration disease, hereditary spastic paraplegia (HSP). To test this, we generated Drosophila (fruitfly) mutants lacking various ER-shaping proteins, and found a range of phenotypes including altered levels of axonal ER, or impaired continuity. We propose two physical properties of axonal ER tubules as critical to its function. First, many local roles of ER do not obviously depend on its physical continuity, but continuity could make axonal ER a long-distance communication channel. Loss of ER continuity in HSP, as in Drosophila mutants, could explain the length-dependence of pathology, since more distal axonal ER would be more prone to separation from the cell body. This model predicts other parameters affecting HSP susceptibility, e.g. axon diameter. Modeling these effects is relevant to other peripheral axonopathies that are major health problems preferentially affect longer axons (e.g. diabetes, chemotherapy). Second, the continuous lumen of ER allows important molecules to diffuse throughout cells, away from the cytosol. Most ER tubules have a diameter around 80-100 nm; however, axonal ER diameter can be as low as 20-30 nm. We propose that axonal ER is designed to limit diffusion through the ER lumen, but we do not know why. Our ultrastructural measurements may provide rich data for modeling this effect, and we are designing experimental strategies to measure it.

34 3D Shoaling Behaviour of Adult ZebrafishLiam O’Shaughnessy, Tatsuo Izawa, Greg Stephens, Josh Shaevitz, Ichiro Masai

Liam O’Shaughnessy Vrije Universiteit Amsterdam

In this work we detail our effort to measure and model social behavior in the swimming dynamics of the adult Zebrafish, Danio rerio. We have constructed a custom tracking apparatus consisting of multiple fast cameras and image-processing software to capture high-resolution, simultaneous dynamics of interacting fish in 3D at the level of posture via marker-less keypoint tracking. Using this apparatus and leveraging the power of the zebrafish system, we aim to quantify the social dynamics of adult zebrafish. We will also connect these dynamics to underlying biological mechanisms through genetic, pharmacological and neural perturbations. In particular we are interested in the transition from a single fish to two fish - how is a system with a single fish different from a system with 2 fish? We also investigate the fighting dynamics between male-male and female-female pairs, and courtship dynamics between male-female pairs.

35 A viscoelastic model predicting cell mechanical memory through mechanical energy dissipation of the cell nucleus Daniel Perez-Calixto, Genaro Vazquez-Victorio, Mathieu Hautefeuille

Daniel Perez-Calixto Universidad Nacional Autónoma de México

Cellular processes are directly impacted by the mechanicals signals from their microenvironment. Some of those signals are directly transmitted from the adhesion complex to the nucleus via the actomyosin apparatus, stressing the organelle and causing reversible and irreversible phenomena. To fully understand the dynamics of this nuclear strain responsible for important mechanoresponsive behaviors, it is crucial to build a model that considers all the nuclear mechanical properties that have been reported to impact nuclear strain. We developed a model integrating the viscoelastic property of the nucleus caused by histone deacetylation and lamins in order to consider the time-dependent contributions of such important nuclear elements. The model managed the contribution of lamin-A,C and lamin-B to the nuclear strain and stress for different ECM stiffnesses as previously shown experimentally by others. Furthermore, the dynamic role of lamins levels is suggested as an explanation of cell mechanical memory through mechanical energy dissipation that induce irreversibility of the nuclear shape. And finally, the model seems to suggest to experimentally study the kinetics of nuclear deformation to better understand ECM-stiffness-related mechanisms on cell nuclei, as the timescale during which stress is applied is of great relevance in defining the future of a cell.

36 Cell shape changes during cellular fate transitionsWolfram Pönisch, Agathe Chaigne, Irene Aspalter, Guillaume Salbreux and Ewa Paluch

Wolfram Pönisch University of Cambridge & University College London

Embryonic development is driven by a series of cellular fate transitions where cells become increasingly specialized. While fate transitions are often accompanied by cell shape changes, how cell shape couples to cell fate is only poorly understood. Here, we present a pipeline to quantify and analyse cell shapes as cells undergo fate transitions. We show how the two- and three-dimensional morphometric features of the cell membrane and nucleus shapes can be quantified, and how the resulting high-dimensional dataset can be analysed with the help of dimensional reduction methods. To identify clusters of cells and classify cells based on those clusters, we use a variety of data mining and machine learning tools. To demonstrate the capabilities of the pipeline, we apply it to study the coupling between cell shape and fate during the exit from naïve pluripotency in mouse embryonic stem cells. We find that cells can be classified into two unambiguously distinguishable clusters: cells with rounded morphology, mostly corresponding to naïve pluripotency, and spread cells, mostly corresponding to cells that have exited naïve pluripotency. The shape changes correlate with the expression of pluripotency markers. After defining the distinct cellular shapes corresponding to distinct cell states, we also obtain the trajectories of morphological features that cells pass during fate transitions. By integrating shape analysis into studies of cellular state changes, we aim to better understand the crosstalk between cellular fate and shape.

37 Mechanical regulation of chemical signalling in the developing brainEva Pillai and Kristian Franze

Eva Pillai University of Cambridge

During nervous system development, growing neurons respond to mechanical as well as chemical signals in their environment. We found that retinal ganglion cell axons grow along stiffness gradients in the developing Xenopus brain. Mechanosensitive ion channels (MSCs) are key players in transducing these mechanical cues into intracellular signals. Pharmacological blocking of MSCs and knockdown of the MSC, Piezo1, caused severe pathfinding errors in vivo. In addition to directly impacting axon growth, downregulation of Piezo1 also dramatically altered the expression of semaphorin3A (Sema3A), a chemical guidance cue known to be critical in axon pathfinding. While Piezo1 knockdown softened brain tissue, knockdown of Sema3A did not alter brain mechanics. Sema3A-producing neuroepithelial cells grown on substrates of varying stiffness adapted expression levels of Sema3A to their mechanical environment. Our results thus indicate that the expression of signalling molecules may be modulated by tissue mechanics, which has important implications given that tissue stiffness changes throughout development as well as during ageing and disease.

38 A helix, a spiral and a toroid: beautiful higher-order structures of DNAKirti Prakash

Kirti Prakash Wellcome-MRC Cambridge Stem Cell Institute

DNA is a long polymer with a double-helix geometry. A helix is probably the most refined structure by which two polymers may couple and provide a pairing mechanism for maximally effective replication. The helix then wraps around octamers of histone proteins to form nucleosomes in a fascinating beads-on-a-string structure. At the final order of compaction, DNA organises itself into globules called chromosome territories. Some outstanding questions in the chromosome biology are: how do 10-nm “bead-on-a-strings” nucleosomes fold into 1000-nm size chromosome territories? Are there intermediary chromatin domains? If so, of what size and shape? And how do they regulate chromatin folding? Does 30-nm chromatin fibre exist? Are chromosomes territories coacervates? We have developed a new method to image DNA in high-resolution and found at least three distinct orders of chromatin states: 30-60 nm (active phase), 120-150 nm (repressed phase) and 250-500 nm (inactive phase). These domains are organised in periodic and symmetric compartments, indicating the spatial organisation of active and inactive regions of the genome. Moreover, we found that, under stress, chromatin dynamically remodels and adapts to a hollow, condensed ring and rod-like configurations, which reverse back to the original structure when stress conditions cease. Here, I propose an alternative classification of higher-order states of DNA based on domain sizes and topological shapes. I also examine the biophysical aspects of DNA condensation and the role of sequence information (both nucleic and protein) in driving the reversible folding of DNA.

39 Exploring force transmission pathways during C elegans proprioceptionRavi Das, Lynn Lin, Iris Ruider, Frederic Català Castro, Nawaphat Malaiwong, Michael Krieg

Iris Ruider ICFO - The Institute of Photonic Sciences

Touching our skin or stretching a leg activates a series of mechanoreceptor neurons that provide us with crucial information about our surrounding and ourselves. The sensation of mechanical force intricately couples our inner with the external world. However, before we become aware, the mechanical information needs to be transmitted to a molecular mechanosensor, independently of how the information is generated. The detailed molecular principle of how mechanical information becomes transmitted between and within cells and eventually activates mechanosensitive ion channels during touch and proprioception is still under debate. Here we employ a CRE/lox based strategy to create cell autonomous defects in cytoskeletal mechanics and identify a role of unc-70 beta spectrin in regulating body posture that is specific to the command interneuron DVA. We also visualize mechanical strain in beta-spectrin during changes in body posture and hypothesize that stress in the cytoskeleton is transferred to a mechanosensitive ion channel to measure the extend of body bending during locomotion. Our data might explain how mechanical properties of neurons enable force transfer mechanics and limit muscle contraction during normal behavior. Since spectrin is highly conserved from worms to humans, our results point towards a conserved mechanism of neuronal stretch signaling of organ volume changes during various physiological functions.

40 Probing the gating mechanism of a mechanosensitive ion channel during gentle touch Neus Sanfeliu, Iris Ruider, Frederic Català , Michael Krieg

Neus Sanfeliu ICFO - The Institute of Photonic Sciences

Mechanosensation is vital for the response of the organism to external stimuli as touch or sound, as well as to other stresses generated inside the body, such as visceral and peripheral proprioception. A failure to sense forces can affect life quality and lead to severe diseases of the nervous system, as neurogenic hypertension or heart failure. Despite the importance of the sense of touch, the molecular basis of the force transmission pathway that translates mechanical stimuli into receptor potentials in vertebrate sensory neurons is poorly understood. Here, we use the model organism Caenorhabditis elegans to study mechanotransduction during gentle body touch. Mechanically gated ion channels (MeT), expressed in touch receptor neurons (TRNs), are proposed, to connect to both the extracellular matrix and the cytoskeleton, but remains unknown whether the MeT channels are activated due to tension transmitted by the cytoskeleton or, by contrast, directly activated by lipid membrane stretch. From touch sensitivity assays, we identified a single point mutation in a conserved protein interaction motif in mec-2 that leads to a complete loss of touch sensation without perturbing transport and localization of MEC-2 inside TRN axons. In addition, wildtype and mutant MEC-2 molecules colocalize with the pore-forming subunit of the MeT channel MEC-4. To elucidate the role of MEC-2 in force transmission, we constructed a MEC-2 force sensor to show that MEC-2 is under mechanical force during body touch. We hypothesize that MEC-2, a conserved human protein stomatin homolog, integrates the MeT complex to the cytoskeleton. Our next goal is to identify the interaction partner of MEC-2 by classical biochemistry and genetic screens.

41 Inferring cell state transition dynamics from pluripotent stem cell heterogeneityLinus Schumacher, Jochen Kursawe, Valerie Wilson, Anestis Tsakiridis, Alexander Fletcher

Linus Schumacher University of Edinburgh

The notion of cell states is increasingly used when classifying cellular behaviour in development, regeneration, and cancer. This is driven in part by a deluge of data comprising snapshots of cell populations at single-cell resolution. Yet quantitative predictive models of cell states and their transitions remain lacking. Such models would allow us to fully leverage datasets to gain a quantitative understanding of cell state transitions; and help to optimise the production of specialised cell types from pluripotent stem cells in vitro. Here, we explore systematically to what extent cell state transition rates can be inferred quantitatively from immunostaining snapshot data. We investigate early cell fate decisions in primitive streak-like populations derived from epiblast stem cells (Tsakiridis et al., 2014). A particular challenge arising from our dataset is that labelling of cell states may be incomplete, i.e., not all of the markers that define a cell state are read out simultaneously in a given experiment. We consider all possible marker combinations as separate cell states and build a minimal mathematical model for the transitions between these states in a growing cell colony. We adopt a Bayesian inference approach to infer cell state transition rates and their uncertainties. We calibrate this model using in vitro data from culture conditions maintaining pluripotency, immunostained for transcription factor expression. With this data-driven modelling approach we identify statistical dependencies between transcription factors indicating regulatory interactions through Bayesian model comparison. We compare models of varying complexity to the available data, and compute each model’s evidence, and can thus incorporate model uncertainty into any predictions. This method is be generally applicable to binary gene expression data from cell populations, and can be extended to analyse single-cell level clonal data.

42 Dynamics of plant root tropism in external electrostatic fieldsNick Oliver, Deniz Tiknaz, Chania Livesey-Clare, Charlie Keizor, Giovanni Sena

Giovanni Sena Imperial College London

Plant roots are able to sense a wide range of physical stimuli in soil, and to respond by modifying their growth direction towards or away the source of the signal (tropism). The orientation of each root tip is a crucial parameter in determining the large-scale architecture of a root system and ultimately the success of its foraging strategy. Root tropisms have been described in response to physical signals such as light, temperature, gravitational field and electrostatic fields. While combinations of physical and molecular mechanisms have been proposed for some root tropisms, to date the response to electric fields has been poorly understood. Here, we will present a systematic quantitative phenomenology of root tropism in electrostatic fields in controlled artificial environments. We will compare the response of various plant species, analyse crucial differences between wild-type and relevant mutants in Arabidopsis, and propose plausible molecular and genetic mechanisms. We will discuss the possible adaptive value for this trait, as well as potential applications for the growing agri-tech sector.

43 Modeling bacterial cell size and shape control under growth perturbationsDiana Serbanescu, Nikola Ojkic, Shiladitya Banerjee

Diana Serbanescu University College London

Bacteria adapt their cell size and molecular resources to environmental conditions in order to optimize their fitness for growth and replication. When subject to growth perturbations, bacteria reallocate their molecular resources to adapt to a new physiological state. While the control of bacterial cell size is well-described by the adder principle at the single-cell level, it remains poorly understood how bacteria allocate their biochemical resources for robust size and shape control. In this study we develop a quantitative biophysical model to predict how the bacterial cell morphologies change under varying nutrient conditions and growth perturbations induced by antibiotics. We show that a balanced tradeoff between the rate of synthesis of division proteins and growth can quantitatively capture experimentally measured cell shape and sizes under different nutrient conditions. Furthermore, our model can predict morphological and biochemical changes in the cell under varying concentrations of ribosome-targeting antibiotics, in agreement with experimental data. Taken together, our model provides a promising alternative to phenomenological growth laws, and can explain the origin of cell size control from biochemical allocation.

44 Force Inference in Heterogeneous Environments and Out-of-Equilibrium States Detection Alexander S. Serov, François Laurent, Christian L. Vestergaard and Jean-Baptiste Masson

Alexander Serov Institut Pasteur

In the following, we introduce two axes of single-molecule-dynamics analysis in a complex environment. We first devise a method to obtain a robust estimate of forces acting on a biomolecule in a heterogeneous environment, with a particular focus on systems modeled by the heterogeneous overdamped Langevin equation. The observed forces include a “spurious” force term due to the heterogeneous diffusivity field. We show how Bayesian inference can be leveraged to reliably infer forces by taking into account various noise sources including such spurious forces. The method is based on the marginalization of the force posterior over all possible spurious force contributions. The approach is combined with a Bayes-factor statistical test for the presence of forces [1]. Furthermore, we investigate ways to identify the breaking of the thermodynamic equilibrium in experimental trajectories. As a simple stationary out-of-equilibrium model, we consider a double-confined dumbbell, the ends of which are exposed to different temperatures. This setup provides a toy model of the motion of a single molecule driven by out-of-equilibrium fluctuations. The energy transfer from the “hot” particle to the “cold” particle can be directly identified in the combined phase space of both particles [2]. However, if only one of the particles is observed - as in a single-molecule experiment - phase-space method is inconclusive. To address the problem, we propose an alternative approach consisting in Bayesian model selection between the models with and without a link to the second particle. These models correspond to the out-of-equilibrium and equilibrium setups, respectively. If the test favors the out-of-equilibrium state, the corresponding posterior distribution can further provide estimates of the direction and the strength of the interaction, as well as of the temperature difference. 1. Serov, A. S. et al. arXiv: 1903.03048 (2019). 2. Battle, C. et al. Science. 352 (6285), 604-607 (2016).

45 Generation of large scale functional human microvascular networks using in vitro mesofluidic assayNegar Shahreza and Emad Moeendarbary

Negar Shahreza University College London

Engineered human tissue models need to be developed to study diseases, develop treatments, and provide a source for replacement of diseased or damaged tissues. However, the inability to vascularise tissue-engineered constructs is a major hindrance which results in limited size and biological complexity. Therefore this study aims to generate functional human microvascular networks on a large scale, using a 3D mesofluidic device. To address this, applying a 3-channel mesofluidic device (23 mm x 3 mm x 1 mm) we co-cultured Green Fluorescent Protein-Human Umbilical Vein Endothelial Cells (GFP-HUVECs) and Normal Human Lung Fibroblasts (NHLFs) embedded within fibrin gel in the vascularization channel. The cells were fed with media through two side channels. By day 7, we formed functional microvascular networks in the vascularisation channel. The integrity of these networks was proven using CD31 staining. CD31 functions as a vascular endothelial cell adhesion molecule and is an endothelial cells marker. Also, to test perfusability we introduced red fluorescent tracer dye (70 kDa) in one of the media channels. Introducing tracer dye demonstrated that the microvascular networks formed were fully functional (97%) on day 7. Altogether, we have established a functional formed vascular network by co-culturing human primary endothelial and fibroblast cells in a mesofluidic platform.

46 The mechanical regulation of Eph/ephrin signalling in the developing brainJana Sipkova, Kristian Franze

Jana Sipkova University of Cambridge

Neuronal development is mediated by chemical as well as mechanical signals. One important group of chemical guidance cues is the membrane-bound ephrins and their receptors, Ephs, which rely on cell-cell contact to mediate downstream signalling. Eph/ephrin signalling is important for many biological processes including retinal axon sorting at the optic tectum during Xenopus retinotectal mapping. However, it is still unknown whether this signalling pathway is also mechanosensitive, and how potential mechanical cues, such as tissue stiffness, could be integrated with Eph/ephrin signalling. Here, in vitro cell cultures on soft substrates and in vivo pathfinding analyses are used to investigate the relationship between mechanical signals and Eph/ephrin signalling during axon guidance.

47 Mechanosensitive junction remodelling promotes robust epithelial morphogenesisMichael F Staddon, Kate E Cavanaugh, Edwin M Munro, Margaret L Gardel, Shiladitya Banerjee

Michael Staddon University College London

Morphogenesis of epithelial tissues requires tight spatiotemporal control of cell shape changes. In vivo, large-scale tissue shape changes are driven by pulsatile contractions of intercellular junctions. The biophysical function of this oscillatory ratchet and its mechanistic basis remain unknown. Here we combine theory and optogenetic experiments to show that mechanosensitive tension remodelling of cell-cell junctions is sufficient to drive large-scale tissue shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but show irreversible deformation under higher magnitudes of contractile stress. The commonly used vertex- based models for epithelium are unable to capture these results, with junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. We thus propose a new model for epithelial cell shape control via strain-dependent remodelling of cell-cell junctional tension. This model is able to capture the viscoelastic behaviour of the epithelial junctions for single contraction pulses of varying timescales, in line with experimental data. The model also captures robust mechanical ratcheting under repeated junctional contractions leading large scale tissue shape changes. We predict that frequency modulation of pulsatile contractions, as opposed to amplitude modulations, will lead to a more efficient mechanical ratchet.

48 Mechanically modulating the nuclear translocation of proteinsAndrew Stannard, Elvira Infante, Sergi Garcia-Manyes

Andrew Stannard King’s College London

Single-molecule force spectroscopy (SMFS) techniques, such as atomic force microscopy, have been widely used to study the mechanical properties of single protein domains by measuring the tensile forces required to unfold them. Using this approach, the mechanical properties of several domains of titin have been studied [1]. In particular, Ig27 (the 27th immunoglobulin-like domain) has become a paradigm and is used as a benchmark of mechanical stability. Here we examine the applicability of these in vitro measurements to an in vivo system - can SMFS aid our understanding of physiological phenomena involving tensile forces? Our experiments investigate whether the nuclear translocation of proteins (passage of cytoplasmic proteins into the nucleus) through the nuclear pore complex (NPC) is regulated by the mechanical stability of the translocating protein. We focus on the translocation of myocardin-related transcription factor A (MRTFA), which localises cytoplasmically in serum-starved cells, but readily translocates to the nucleus upon serum stimulation [2]. With MRTFA fluorescently tagged, its nuclear translocation can be initiated, monitored, quantified, and modelled to extract translocation rate constants. Using this model system, we systematically modify the mechanical stability of fluorescently-tagged MRTFA constructs via the inclusion of domains with varying mechanical stabilities that are well characterised via SMFS. In particular, we concatenate MRTFA-GFP with the wild type and point mutations of Ig27, exhibiting a spectrum of mechanical stabilities in vitro. We find that the rate constant associated with nuclear import displays a clear inverse correlation with the mechanical stability of the translocating construct, demonstrating the mechanoselectivity of the NPC [3]. [1] H. Li et al., Nature 418, 998 (2002) [2] C. Ho et al., Nature 497, 507 (2013) [3] A. Stannard*, E. Infante* et al., Nat. Phys. (in press)

49 Flows and Winches: Closing Wounds in 3 DimensionsRobert J Tetley, Michael F Staddon, Shiladitya Banerjee, Yanlan Mao

Robert Tetley MRC Laboratory for Molecular Cell Biology, UCL

The behaviours of epithelial cells and tissues are, in part, determined by their mechanical properties. However, the contribution of tissue mechanics to wound healing remains poorly understood. We have investigated the role of tissue mechanics during wound healing using the wing imaginal disc of Drosophila. Through a multidisciplinary approach combining fly genetics, live imaging, quantitative 4D image analysis, biophysical perturbations and computational modelling, we have demonstrated that the 3-dimensional mechanical landscape of the tissue is crucial for efficient wound closure. Within the plane of the epithelium, wound healing is punctuated by cell-cell intercalation events at the wound edge, where a contractile actomyosin purse string forms. These intercalations allow cells to flow into the centre of the wound like molecules in a liquid. We find that tissue fluidity can be enhanced through a decrease in tissue contractility, resulting in an acceleration of wound closure despite a weakened purse string. Orthogonal to the epithelial plane, cells unexpectedly shorten dramatically along their apicobasal axis as wound closure progresses. This reduction in apicobasal height is driven by myosin cables lining the lateral membranes of wound edge cells, which increase wound edge apicobasal tension. We find that the co-ordinated mechanical activity of the apical actomyosin purse string and lateral myosin cables is required for efficient wound closure. Furthermore, we find that increased lateral tension positively feeds back on the purse string to stabilise reductions in wound area.

50 Differential Growth rates determine positioning of folding in complex epitheliaMelda Tozluoglu, Maria Duda, Natalie Kirkland, Ricardo Barrientos, Jemima Burden, Jose Munoz, Yanlan Mao

Melda Tozluoglu University College London

Understanding the regulation of tissue growth is of significant clinical interest, and can shed light on processes ranging from tissue regeneration to inhibition of uncontrolled growth in cancer. Although the roles of biochemical regulatory mechanisms of tissue growth and shape have been key subjects of research, our understanding of the roles for mechanical properties of tissues is still limited. This imbalance stems from the difficulties in investigating mechanical roles of proteins, such as in generating tension, independently from their biochemical functions in experiments. In our computational approach, we utilise a finite element methodology to answer how the interplay between the differential growth rates, physical properties of the cells, and of the extracellular matrix (ECM), lead to the final tissue architecture. The model system we use is the wing imaginal disc of Drosophila Melanogaster. Starting from heterogeneities in growth and physical properties, we try to identify the minimum set of requirements that drives the three-dimensional folded structure of the wing disc. We identify that the early differential growth rates are the main driver for the specific locations of fold initiation. In turn, stiffness differences between structurally different layers of the cells, coupled with the resistances of both apical and basal ECM to the growing tissue, drive the fold initiation. These physical property heterogeneities and confinement effects influence fold positions to a lesser degree. Using our computational model, we can successfully predict the emergent fold morphology of mutants in vivo, via perturbations solely on differential growth rates in silico.

51 Cell Matrix Adhesions and Collective Cell Migration in vivo: The role of X-Chef-1Melissa Turan, Elias H Barriga, Roberto Mayor

Melissa Turan University College London

Collective cell migration (CCM) is involved in several developmental and physiological processes. During collective cell migration, in addition to molecular signals, cells interact with mechanical cues from their environment. This study focuses on the collective migration of the Xenopus laevis cephalic neural crest, where it was recently demonstrated that stiffening of the substrate is required to trigger an epithelial to mesenchymal transition (EMT), and the subsequent onset of migration. However, the molecular mechanism by which the neural crest transduces mechanical cues from its substrate remains unclear. Here, we investigate the role of Crk associated substrate protein, X-chef-1 during neural crest migration. This scaffolding protein within the integrin signaling pathway was shown to be specifically expressed in the neural crest prior to the onset of migration. We find that X-chef-1 localizes to focal adhesions and promotes migration through the stabilization of cell matrix adhesions and the force dependent activation of polarity effector Rac-1. Taken together, our results suggest that X-chef-1 plays a role in coordinating the response of the neural crest to its substrate during migration.

52 Role of Actin Crosslinkers in Cortex Tension RegulationNeza Vadnjal, Murielle Serres, Genevieve Lavoie, Philippe Roux, Ricardo Henriques, Ewa Paluch

Neza Vadnjal MRC Laboratory for Molecular Cell Biology, UCL

Animal cell shape changes during processes such as division are controlled by the cellular cortex, a thin network underlying the plasma membrane composed of actin, myosin, and other actin binding molecules. Changes in cortex tension result in cell shape changes. Most studies of tension regulation have focused on the role of myosin activity, while overlooking the impact of the organisation of the actin network itself. Particularly, actin filament length and some crosslinkers have been shown to be involved in tension regulation. To determine which actin crosslinkers are most important in tension regulation we analysed cortex-enriched blebs isolated from interphase and mitotic cells by mass spectometry. The identified crosslinkers have a range of different sizes (5-100 nm). Size of actin binding proteins can affect their localization due to steric effects. To determine how the size of actin crosslinkers affects their localization within actin cortex, we started optimizing dSTORM and expansion microscopy for multi-colour super-resolution visualization of actin cortex.

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