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.Cell shape emerges from the interaction of many constituent elements-notably, the cytoskeleton, the cell membrane and cellsubstrate adhesions-that have been studied in great detail at the molecular level 1-3 ; however, the mechanism by which global morphology is generated and maintained at the cellular scale is not understood. Many studies have characterized the morphological effects of perturbing various cytoskeletal and other cellular components (for example, ref. 4); yet, there have been no comprehensive efforts to try to understand cell shape from first principles. Here we address this issue in the context of motile epithelial keratocytes derived from fish skin. Fish keratocytes are among the fastest moving animal cells, and their motility machinery is characterized by extremely rapid molecular dynamics and turnover [5][6][7][8] . At the same time, keratocytes are able to maintain nearly constant speed and direction during movement over many cell lengths. Their shapes, consisting of a bulbous cell body at the rear attached to a broad, thin lamellipodium at the front and sides, are simple, stereotyped and notoriously temporally persistent 9,10 . The molecular dynamism of these cells, combined with the persistence of their global shape and behaviour, make them an ideal model system for investigating the mechanisms of cell shape determination.The relative simplicity of keratocytes has inspired extensive experimental and theoretical investigations into this cell type 5-17 , considerably advancing the understanding of cell motility. A notable example is the graded radial extension (GRE) model 12 , which was an early attempt to link the mechanism of motility at the molecular level with overall cell geometry. The GRE model proposed that local cell extension (either protrusion or retraction) occurs perpendicular to the cell edge, and that the magnitude of this extension is graded from a maximum near the cell midline to a minimum towards the sides. Although this phenomenological model has been shown experimentally ...

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton1: protrusion of the leading edge requires assembly of a network of actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly3, it is not clear how activity of these locally acting proteins could be coordinated over the whole-cell distance scale. Here we show that nonmuscle myosin II plays a direct role in actin network disassembly in crawling cells. In moving fish keratocytes, 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.

Cells are dynamic systems capable of spontaneously switching among stable states. One striking example of this is spontaneous symmetry breaking and motility initiation in fish epithelial keratocytes. Although the biochemical and mechanical mechanisms that control steady-state migration in these cells have been well characterized, the mechanisms underlying symmetry breaking are less well understood. In this work, we have combined experimental manipulations of cell−substrate adhesion strength and myosin activity, traction force measurements, and mathematical modeling to develop a comprehensive mechanical model for symmetry breaking and motility initiation in fish epithelial keratocytes. Our results suggest that stochastic fluctuations in adhesion strength and myosin localization drive actin network flow rates in the prospective cell rear above a critical threshold. Above this threshold, high actin flow rates induce a nonlinear switch in adhesion strength, locally switching adhesions from gripping to slipping and further accelerating actin flow in the prospective cell rear, resulting in rear retraction and motility initiation. We further show, both experimentally and with model simulations, that the global levels of adhesion strength and myosin activity control the stability of the stationary state: The frequency of symmetry breaking decreases with increasing adhesion strength and increases with increasing myosin contraction. Thus, the relative strengths of two opposing mechanical forcescontractility and cell−substrate adhesion-determine the likelihood of spontaneous symmetry breaking and motility initiation.symmetry breaking | cell migration | adhesion | myosin S tationary adherent cells are symmetric systems, in which all forces generated by and acting on the cell are balanced, allowing the cell to maintain a consistent shape and position. In order for a stationary cell to initiate motility, the symmetry of the system must first be broken by external or internal cues. Mechanisms of symmetry breaking have been extensively studied in chemotactic cells, such as neutrophils and Dictyostelium, which break symmetry and migrate in the direction of chemoattractant gradients (1). However, stationary neutrophils also break symmetry and initiate motility in uniform baths of chemoattractant (2, 3), indicating that a directional cue is not required. In addition, nonchemotactic cells, including fish epithelial keratocytes and keratocyte fragments, are able to break symmetry and initiate motility in the absence of external cues (4, 5). Thus, while external cues may confer a preferred directionality, intrinsic cellular instabilities can be sufficient for symmetry breaking. (8) and polarization and symmetry breaking in budding yeast (9). Mechanical instabilities can also drive symmetry breaking and have been shown to be particularly relevant for force-generating cytoskeletal systems (10-12). Stochastic fluctuations in actin filament densities and mechanical feedback between motor proteins and cytoskeletal elements can drive symm...