Teasing apart the impact of electric fields on single-cell motility
Cell motility in response to environmental cues forms the basis of many developmental processes in multicellular organisms. One such environmental cue is an electric field, which induces a form of motility known as electrotaxis. Electrotaxis has evolved in a number of cell types to guide wound healing, and has been associated with different cellular processes, suggesting that observed electrotactic behaviour is likely a combination of multiple distinct effects arising from the presence of an electric field. In order to determine the different mechanisms by which observed electrotactic behaviours emerge, and thus to design electric fields that can be applied to direct and control electrotaxis, we require accurate quantitative predictions of cellular responses to externally-applied fields. Here, we use mathematical modelling to formulate and parametrise a variety of hypothetical descriptions of how cell motility may change in response to an electric field. We calibrate our model to observed data using synthetic likelihoods and Bayesian sequential learning techniques, and demonstrate that electric fields impact cellular motility in three distinct ways.
Mechanobiological control of immune cell activation
New perspective of mechanobiology is currently emerging across multiple disciplines in the biomedical sciences. In contrast to conventional believes, recent evidence indicates that cells regulate their cell mechanics not downstream of signalling events triggered by ligand–receptor binding, but that cells may employ feedback mechanisms to dynamically adjust their mechanics in response to external stimuli. Quantifying cellular forces has therefore become an contentious challenge across multiple disciplines at the interface of biophysics, cell-biology, and immunology. Mechanical forces are especially important for the activation of immune T cells. Using a suite of advanced quantitative super-resolution imaging and force probing methodologies to analyse resting and activated T cells, we demonstrate activating T cells sequentially rearrange their nanoscale mechanobiology, creating a previously unreported ramifying actin network above the immunological synapse (IS). We show evidence that the kinetics of the antigen engaging the T-cell receptor controls the nanoscale actin organisation and mechanics of the IS. Using an engineered T-cell system expressing a specific T-cell receptor and stimulated by a range of antigens, force measurements revealed that the peak force experienced by the T-cell receptor during activation was independent of the kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the IS revealed a striking dependence on the antigen kinetics. Taken together, these findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalise the force experienced by the T-cell receptor in an antigen specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the length- and time-scale of T-cell receptor signalling.
Ratchets in body morphogenesis
Work from the past 15 years has highlighted that organ morphogenesis occurs through discrete force-generating events. In contrast to tissues that have been thoroughly investigated in Drosophila and zebrafish, which are generally mostly flat, C. elegans embryos resemble a tube and its elongation does not involve non-muscle actomyosin pulses. Instead, the 2nd phase of its elongation relies on repeated muscle contractions. By combining various microscopy modes, we found that the asynchronous contractions of muscles represent a source of planar polarity that helps extend anterior-posterior adherens junctions in the epidermis. In particular, localized muscle contractions cause the embryo to rotate within its eggshell causing junctions between epidermal cells to repeatedly become under tension. In turn, this promotes the fusion of E-cadherin vesicles with junctions each time they are under tension. Together with our recent findings about the muscle-promoted shortening of actin bundles in the dorso-ventral epidermis (Lardennois et al, Nature 2019), these findings provide a ratchet mechanism for C. elegans embryonic elongation.
Active nematic behaviours of cellular monolayers
I will present how active nematic activity of cellular monolayers can help to understand biological processes and tissue organization. In the first part, I will show how these active behaviours and stresses govern cell extrusion. By modelling the epithelium as an active nematic liquid crystal and measuring mechanical parameters such as strain rates and stresses measurements within cellular monolayers, we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. The results highlight the importance of active nematic nature of epithelia. In the second part, I will focus on the intriguing extensile behaviour of epithelial cells as a collective when single cells behave as contractile systems. Through a combination of experiments and in silico modelling, we uncover the mechanism behind this switch of behaviour of cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the build up of tension at the cell-substrate interface through an increase in actin stress fibres and higher traction forces. Such differences in extensivity and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation.
The cytoskeleton steering search processes
Immune cells require to move efficiently in order to find pathogens and protect their host. But how does cellular search become efficient? In my talk I will introduce immune cell migration and methods to quantify it, explain ways of cells to switch between persistent and diffuse migration, describe the universal coupling law between speed and persistence, show the role which this coupling plays in the efficiency of search and end by suggesting ways of perturbing it.
How do tissue cells sense their “personal space”?
Like people, cells in the human body are territorial about their personal space. They seem to know how much space they like, and if things get too tight, most cells prefer to leave than stay. This simple logic underlies complex cell decisions during tissue building, immune responses, and wound healing. Here, we found that this logic works only when cells are squeezed to the degree at which their largest component – the nucleus – gets physically stretched to signal the squeeze in a mechanochemical fashion. We therefore propose that the nucleus functions as a ruler allowing living cells to measure their personal space.
Coping with Mechanical Stress: Tissue dynamics in development and repair
During growth and development, tissue dynamics, such as tissue folding, cell intercalations and oriented cell divisions, are critical for shaping tissues and organs. However, less is known about how tissues regulate their dynamics during tissue homeostasis and repair, to maintain their shape after development. In this talk, we will discuss how differential growth rates can generate precise folds in tissues. We will also discuss how tissues respond to mechanical perturbations, such as stretching or wounding, by altering their actomyosin contractile structures, to change tissue dynamics, and thus preserve tissue shape and patterning. We combine genetics, biophysics and computational modelling to study these processes.
Spatio-temporal control of microtubule mechanics during epithelial morphogenesis
Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, our knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. To unravel the origin and control of microtubule-based forces during tissue formation, maintenance and disease, we use novel optical and chemical tools in conjunction with classical genetic approaches in the Drosophila wing, a well-established and powerful in vivo system for the systematic characterization of force-control.
Dynamic coupling of cell-cell signaling, force generation and tissue remodeling during neural tube closure
A fundamental challenge is to understand how the collective dynamics of cells that shape tissues emerge through the dynamic interplay of gene expression, cellular signalling and force generation. In this talk I will summarize our efforts to address this challenge in the context of neural tube closure in ascidian embryos. During the last step of neural tube closure, known as zippering, the neural folds meet and fuse in a posterior to anterior sequence to separate the closed neural tube from the overlying epidermis. Sequential activation of RhoA and Myosin II on neural-epidermal (Ne/Epi) junctions just ahead of the advancing zipper drive their rapid contraction. Tissue level asymmetries in contractility (high on Ne/Epi junctions ahead of the zipper; low on Ne/Ne and Epi/Epi junctions behind the zipper) and dissipation of tissue resistance behind the zipper through junctional exchange (Ne/Epi -> Ne/Ne and Epi/Epi) creates a force balance that converts local junctions into directional zipper movement. Finally, dynamic signalling across the Ne/Epi boundary, and across the midline between neural multiple close tissue-level feedback loops that sustain tissue level contractile asymmetry and propagate the wave of sequential junction contractions.
Mechanical forces in the developing lung
The morphogenetic patterning that generates three-dimensional (3D) tissues requires dynamic concerted rearrangements of individual cells with respect to each other. We have developed microfluidic approaches to investigate the mechanical forces and downstream cellular rearrangements responsible for generating the airways of the lung. I will discuss how we combine these experimental techniques with computational models to uncover the physical forces that drive development of complex epithelial geometries. I will also describe efforts to uncover and actuate the different physical mechanisms used to build the airways in lungs from birds, mammals, and reptiles.
On the control of epithelial mechanics during morphogenesis: How to seal epithelial gaps
During embryogenesis, dramatic tissue reorganization occurs under the control of specific signaling pathways. Mechanical forces are coordinated in order to rearrange biological tissues and shape organs. The mechanisms at the origin of the generation and regulation of these forces during development to ensure correct cell positioning and tissue shaping remain elusive. Using Drosophila as a model, my group is interested in understanding the principles underlying the control of fundamental modes of tissue remodeling such as epithelial contraction, tissue spreading or folding during development. Here, I will present recent works on the generation and regulation of forces during tissue contraction during Drosophila dorsal closure. I will also discuss the mechanisms controlling cell/cell junctional length and tension and ensuring scaling during epithelial contraction. I will eventually present new results on the mechanisms controlling epithelial sealing and body segment assembly.
Mechanobiology of the mitotic spindle
At the onset of division the cell forms a spindle, a micro-machine made of microtubules, which divide the chromosomes by pulling on kinetochores, protein complexes on the chromosome. The central question in the field is how accurate chromosome segregation results from the interactions between kinetochores, microtubules and the associated proteins. Because the spindle is basically a mechanical apparatus, the understanding of its functioning requires techniques based on mechanical perturbations. By using laser cutting of microtubules, we have shown that a bundle of antiparallel microtubules, termed “bridging fibre”, connects sister kinetochore fibres in human spindles. Bridging microtubules are linked together by the protein regulator of cytokinesis 1 (PRC1). To explore the role of bridging fibres in chromosome movements, we developed an optogenetic approach to remove PRC1 from the spindle to the plasma membrane in a fast and reversible manner by using light. These experiments showed that bridging fibres promote chromosome alignment at the metaphase plate by forces that depend on the microtubule overlap within the bridging fibre. We then developed a speckle microscopy assay to measure the poleward flux of individual spindle microtubules and found that the flux of bridging microtubules is faster than that of kinetochore microtubules, suggesting that the lateral length-dependent forces that the bridging fibre exerts onto kinetochore fibres drive the sliding of kinetochore fibres along the bridging fibre, thereby helping to centre the chromosomes on the spindle. By combining a theoretical model with super resolution imaging of the bridging fibres, we discovered that they are twisted in the shape of a left-handed helix, making the spindle a chiral object. This finding suggests that torques exist in the spindle in addition to linear (pushing and pulling) forces. During anaphase, bridging microtubules promote chromosome segregation by sliding apart, which is driven by the motor activity of kinesin-4 and kinesin-5. This sliding pushes the attached kinetochore fibres and kinetochores poleward to segregate chromosomes. Understanding the role of bridging microtubules in force generation and chromosome movements not only sheds light on the mechanobiology of a well-functioning spindle, but will also help to understand the origins of errors in chromosome segregation.