Effective membrane tension: A long-range integrator of cellular dynamics.

Membrane tension has been proposed to mechanically couple processes along the cell's boundary. In this issue of Cell, De Belly et al. show that local protrusion or contraction elicit a global membrane tension increase within seconds, whereas tension perturbations that engage only the membrane remain localized.

Mechanical Patterning in Animal Morphogenesis.

Morphogenesis is one of the most remarkable examples of biological pattern formation. Despite substantial progress in the field, we still do not understand the organizational principles responsible for the robust convergence of the morphogenesis process across scales to form viable organisms under variable conditions. Achieving large-scale coordination requires feedback between mechanical and biochemical processes, spanning all levels of organization and relating the emerging patterns with the mechanisms driving their formation. In this review, we highlight the role of mechanics in the patterning process, emphasizing the active and synergistic manner in which mechanical processes participate in developmental patterning rather than merely following a program set by biochemical signals. We discuss the value of applying a coarse-grained approach that considers the large-scale dynamics and feedback and complements the reductionist approach focused on molecular detail. A central challenge in this approach is identifying relevant coarse-grained variables and developing effective theories that can serve as a basis for an integrated framework toward understanding this remarkable pattern-formation process.

Load Adaptation of Lamellipodial Actin Networks.

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Experiment, theory, and the keratocyte: An ode to a simple model for cell motility.

Lamellipodial locomotion of fish keratocytes is one of the simplest examples of actin-based motility. In the last four decades, fruitful collaborations between experimentalists and theorists have resulted in a detailed mechanistic understanding of the self-organized lamellipodial engine powering keratocyte motility. Here we review the mechanical mechanisms underlying keratocyte migration, highlighting the interplay between modeling and experiments that led to insights regarding the dynamics of actin network organization, cell shape, and self-polarization. We discuss how to apply lessons learnt from keratocytes to understand cell migration in more complex, physiological contexts.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Hydra Regeneration: Closing the Loop with Mechanical Processes in Morphogenesis.

The convergence of morphogenesis into viable organisms under variable conditions suggests closed-loop dynamics involving multiscale functional feedback. We develop the idea that morphogenesis is based on synergy between mechanical and bio-signaling processes, spanning all levels of organization: molecular, cellular, tissue, up to the whole organism. This synergy provides feedback within and between all levels of organization, to close the loop between the dynamics of the morphogenesis process and its robust functional outcome. Hydra offer a powerful platform to explore this direction, thanks to their simple body plan, extraordinary regeneration capabilities, and the accessibility and flexibility of their tissues. Our recent experiments show that structural inheritance of the actomyosin organization directs body-axis formation during Hydra regeneration. Morphogenesis is then stabilized through dynamic cytoskeletal reorganization induced by the inherited structure. The observed cytoskeletal stability and reorganization capabilities suggest that mechanical feedback integrates with biochemical processes to establish viable patterns and ensure canalization.

Myosin II contributes to cell-scale actin network treadmilling through network disassembly.

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton: protrusion of the leading edge requires assembly of a network of actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

Front-to-rear membrane tension gradient in rapidly moving cells.

Membrane tension is becoming recognized as an important mechanical regulator of motile cell behavior. Although membrane-tension measurements have been performed in various cell types, the tension distribution along the plasma membrane of motile cells has been largely unexplored. Here, we present an experimental study of the distribution of tension in the plasma membrane of rapidly moving fish epithelial keratocytes. We find that during steady movement the apparent membrane tension is ∼30% higher at the leading edge than at the trailing edge. Similar tension differences between the front and the rear of the cell are found in keratocyte fragments that lack a cell body. This front-to-rear tension variation likely reflects a tension gradient developed in the plasma membrane along the direction of movement due to viscous friction between the membrane and the cytoskeleton-attached protein anchors embedded in the membrane matrix. Theoretical modeling allows us to estimate the area density of these membrane anchors. Overall, our results indicate that even though membrane tension equilibrates rapidly and mechanically couples local boundary dynamics over cellular scales, steady-state variations in tension can exist in the plasma membranes of moving cells.

Theoretical analysis of membrane tension in moving cells.

Lateral tension in cell plasma membranes plays an essential role in regulation of a number of membrane-related intracellular processes and cell motion. Understanding the physical factors generating the lateral tension and quantitative determination of the tension distribution along the cell membrane is an emerging topic of cell biophysics. Although experimental data are accumulating on membrane tension values in several cell types, the tension distribution along the membranes of moving cells remains largely unexplored. Here we suggest and analyze a theoretical model predicting the tension distribution along the membrane of a cell crawling on a flat substrate. We consider the tension to be generated by the force of actin network polymerization against the membrane at the cell leading edge. The three major factors determining the tension distribution are the membrane interaction with anchors connecting the actin network to the lipid bilayer, the membrane interaction with cell adhesions, and the force developing at the rear boundary due to the detachment of the remaining cell adhesion from the substrate in the course of cell crawling. Our model recovers the experimentally measured values of the tension in fish keratocytes and their dependence on the number of adhesions. The model predicts, quantitatively, the tension distribution between the leading and rear membrane edges as a function of the area fractions of the anchors and the adhesions.

An adhesion-dependent switch between mechanisms that determine motile cell shape.

Keratocytes are fast-moving cells in which adhesion dynamics are tightly coupled to the actin polymerization motor that drives migration, resulting in highly coordinated cell movement. We have found that modifying the adhesive properties of the underlying substrate has a dramatic effect on keratocyte morphology. Cells crawling at intermediate adhesion strengths resembled stereotypical keratocytes, characterized by a broad, fan-shaped lamellipodium, clearly defined leading and trailing edges, and persistent rates of protrusion and retraction. Cells at low adhesion strength were small and round with highly variable protrusion and retraction rates, and cells at high adhesion strength were large and asymmetrical and, strikingly, exhibited traveling waves of protrusion. To elucidate the mechanisms by which adhesion strength determines cell behavior, we examined the organization of adhesions, myosin II, and the actin network in keratocytes migrating on substrates with different adhesion strengths. On the whole, our results are consistent with a quantitative physical model in which keratocyte shape and migratory behavior emerge from the self-organization of actin, adhesions, and myosin, and quantitative changes in either adhesion strength or myosin contraction can switch keratocytes among qualitatively distinct migration regimes.

Mechanism of shape determination in motile cells.

The shape of motile cells is determined by many dynamic processes spanning several orders of magnitude in space and time, from local polymerization of actin monomers at subsecond timescales to global, cell-scale geometry that may persist for hours. Understanding the mechanism of shape determination in cells has proved to be extremely challenging due to the numerous components involved and the complexity of their interactions. Here we harness the natural phenotypic variability in a large population of motile epithelial keratocytes from fish (Hypsophrys nicaraguensis) to reveal mechanisms of shape determination. We find that the cells inhabit a low-dimensional, highly correlated spectrum of possible functional states. We further show that a model of actin network treadmilling in an inextensible membrane bag can quantitatively recapitulate this spectrum and predict both cell shape and speed. Our model provides a simple biochemical and biophysical basis for the observed morphology and behaviour of motile cells.

Actin disassembly clock determines shape and speed of lamellipodial fragments.

A central challenge in motility research is to quantitatively understand how numerous molecular building blocks self-organize to achieve coherent shape and movement on cellular scales. A classic example of such self-organization is lamellipodial motility in which forward translocation is driven by a treadmilling actin network. Actin polymerization has been shown to be mechanically restrained by membrane tension in the lamellipodium. However, it remains unclear how membrane tension is determined, what is responsible for retraction and shaping of the rear boundary, and overall how actin-driven protrusion at the front is coordinated with retraction at the rear. To answer these questions, we utilize lamellipodial fragments from fish epithelial keratocytes which lack a cell body but retain the ability to crawl. The absence of the voluminous cell body in fragments simplifies the relation between lamellipodial geometry and cytoskeletal dynamics. We find that shape and speed are highly correlated over time within individual fragments, whereby faster crawling is accompanied by larger front-to-rear lamellipodial length. Furthermore, we find that the actin network density decays exponentially from front-to-rear indicating a constant net disassembly rate. These findings lead us to a simple hypothesis of a disassembly clock mechanism in which rear position is determined by where the actin network has disassembled enough for membrane tension to crush it and haul it forward. This model allows us to directly relate membrane tension with actin assembly and disassembly dynamics and elucidate the role of the cell membrane as a global mechanical regulator which coordinates protrusion and retraction.

Canalized Morphogenesis Driven by Inherited Tissue Asymmetries in Hydra Regeneration.

The emergence and stabilization of a body axis is a major step in animal morphogenesis, determining the symmetry of the body plan as well as its polarity. To advance our understanding of the emergence of body axis polarity, we study regenerating Hydra. Axis polarity is strongly memorized in Hydra regeneration even in small tissue segments. What type of processes confer this memory? To gain insight into the emerging polarity, we utilize frustrating initial conditions by studying regenerating tissue strips which fold into hollow spheroids by adhering their distal ends of opposite original polarities. Despite the convoluted folding process and the tissue rearrangements during regeneration, these tissue strips develop in a reproducible manner, preserving the original polarity and yielding an ordered body plan. These observations suggest that the integration of mechanical and biochemical processes supported by their mutual feedback attracts the tissue dynamics towards a well-defined developmental trajectory biased by weak inherited cues from the parent animal. Hydra thus provide an example of dynamic canalization in which the dynamic rules are instilled, but, in contrast to the classical picture, the detailed developmental trajectory does not unfold in a programmatic manner.

Plasticity of body axis polarity in Hydra regeneration under constraints.

One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to explore the plasticity of polarity determination during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the polarity and original positions of the rings along the body axes of the parent animals. Under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. Importantly, the site of head regeneration exhibits a strong bias toward the edges of the tissue, even when this involves polarity reversal. In particular, we observe head formation at an originally aboral tissue edge, which is not compatible with models of Hydra regeneration based only on preexisting morphogen gradients or an injury response. The site of the new head invariably contains an aster-like defect in the organization of the supra-cellular ectodermal actin fibers. While a defect is neither required nor sufficient for head formation, we show that the defect at the new head site can arise via different routes, either appearing directly following excision as the tissue seals at its edge or through de novo defect formation at the fusion site. Altogether, our results show that the emergence of a polar body axis depends on the original polarity and position of the excised tissues as well as structural factors, suggesting that axis determination is an integrated process that arises from the dynamic interplay of multiple biochemical and mechanical processes.

Cell motility: the integrating role of the plasma membrane.

The plasma membrane is of central importance in the motility process. It defines the boundary separating the intracellular and extracellular environments, and mediates the interactions between a motile cell and its environment. Furthermore, the membrane serves as a dynamic platform for localization of various components which actively participate in all aspects of the motility process, including force generation, adhesion, signaling, and regulation. Membrane transport between internal membranes and the plasma membrane, and in particular polarized membrane transport, facilitates continuous reorganization of the plasma membrane and is thought to be involved in maintaining polarity and recycling of essential components in some motile cell types. Beyond its biochemical composition, the mechanical characteristics of the plasma membrane and, in particular, membrane tension are of central importance in cell motility; membrane tension affects the rates of all the processes which involve membrane deformation including edge extension, endocytosis, and exocytosis. Most importantly, the mechanical characteristics of the membrane and its biochemical composition are tightly intertwined; membrane tension and local curvature are largely determined by the biochemical composition of the membrane and the biochemical reactions taking place; at the same time, curvature and tension affect the localization of components and reaction rates. This review focuses on this dynamic interplay and the feedbacks between the biochemical and biophysical characteristics of the membrane and their effects on cell movement. New insight on these will be crucial for understanding the motility process.

Cytoskeletal symmetry breaking in animal cells.

Symmetry breaking is a crucial step in structure formation and function of all cells, necessary for cell movement, cell division, and polarity establishment. Although the mechanisms of symmetry breaking are diverse, they often share common characteristics. Here we review examples of nematic, polar, and chiral cytoskeletal symmetry breaking in animal cells, and analogous processes in simplified reconstituted systems. We discuss the origins of symmetry breaking, which can arise spontaneously, or involve amplification of a pre-existing external or internal bias to the whole cell level. The underlying mechanisms often involve both chemical and mechanical processes that cooperate to break symmetry in a robust manner, and typically depend on the shape, size, or properties of the cell's boundary.

Centering and symmetry breaking in confined contracting actomyosin networks.

Centering and decentering of cellular components is essential for internal organization of cells and their ability to perform basic cellular functions such as division and motility. How cells achieve proper localization of their organelles is still not well-understood, especially in large cells such as oocytes. Here, we study actin-based positioning mechanisms in artificial cells with persistently contracting actomyosin networks, generated by encapsulating cytoplasmic Xenopus egg extracts into cell-sized 'water-in-oil' droplets. We observe size-dependent localization of the contraction center, with a symmetric configuration in larger cells and a polar one in smaller cells. Centering is achieved via a hydrodynamic mechanism based on Darcy friction between the contracting network and the surrounding cytoplasm. During symmetry breaking, transient attachments to the cell boundary drive the contraction center to a polar location. The centering mechanism is cell-cycle dependent and weakens considerably during interphase. Our findings demonstrate a robust, yet tunable, mechanism for subcellular localization.

In order to survive, cells need to react to their environment and change their shape or the localization of their internal components. For example, the nucleus ­ the compartment that contains the genetic information ­ is often localized at the center of the cell, but it can also be positioned at the side, for instance when cells move or divide asymmetrically. Cells use multiple positioning mechanisms to move their internal components, including a process that relies on networks of filaments made of a protein known as actin. These networks are constantly remodeled as actin proteins are added and removed from the network. Embedded molecular motors can cause the network of actin filaments to contract and push or pull on the compartments. Yet, the exact way these networks localize components in the cell remains unclear, especially in eggs and other large cells. To investigate this question, Ierushalmi et al. studied the actin networks in artificial cells that they created by enclosing the contents of frog eggs in small droplets surrounded by oil. This showed that the networks contracted either to the center of the cell or to its side. Friction between the contracting actin network and the fluid in the cell generated a force that tends to push the contraction center towards the middle of the cell. In larger cells, this led to the centering of the actin network. In smaller cells however, the network transiently attached to the boundary of the cell, leading the contraction center to be pulled to one side. By developing simpler artificial cells that mimic the positioning processes seen in real-life cells, Ierushalmi et al. discovered new mechanisms for how cells may center or de-center their components. This knowledge may be useful to understand diseases that can emerge when the nucleus or other compartments fail to move to the right location, and which are associated with certain organs developing incorrectly.

Cellular Organization: Bulk Actin Network Flows Drive Ooplasm Segregation.

Actin networks in the bulk cytoplasm, rather than cortical dynamics, drive ooplasm segregation in zebrafish oocytes. A contracting actin network drags the ooplasm toward the animal pole, while 'comet tails' push the yolk granules in the opposite direction.