Erin L. Barnhart

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.

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.

Actin–myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility

We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin–myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.

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.

Membrane Tension in Rapidly Moving Cells Is Determined by Cytoskeletal Forces

BACKGROUND Membrane tension plays an essential role in cell motility. The load imposed by the tensed membrane restrains actin polymerization, promotes rear retraction, and influences membrane transport. Moreover, membrane tension is crucial for large-scale coordination of cell boundary dynamics. Despite its importance, little is known about how membrane tension is set and regulated in cells. The prevailing hypothesis is that membrane tension is largely controlled by membrane-cytoskeleton adhesion and/or changes in membrane area. RESULTS In this work, we measure the apparent membrane tension in rapidly moving fish epithelial keratocytes under normal and perturbed conditions with a tether-pulling assay. We find that enlargement of the cell surface area by fusion with giant unilamellar vesicles (GUVs) has only minor effects on membrane tension and on cell movement. However, modulation of the cytoskeletal forces has a substantial influence on tension: reduction of the actin-pushing forces along the cells leading edge leads to a significant decrease in membrane tension, whereas increase of the strength of adhesion and/or decrease of myosin-induced contraction leads to higher tension. CONCLUSIONS We find that the membrane tension in rapidly moving keratocytes is primarily determined by a mechanical force balance between the cell membrane and cytoskeletal forces. Our results highlight the role of membrane tension as a global mechanical regulator of cell behavior.

Bipedal Locomotion in Crawling Cells

Many complex cellular processes from mitosis to cell motility depend on the ability of the cytoskeleton to generate force. Force-generating systems that act on elastic cytoskeletal elements are prone to oscillating instabilities. In this work, we have measured spontaneous shape and movement oscillations in motile fish epithelial keratocytes. In persistently polarized, fan-shaped cells, retraction of the trailing edge on one side of the cell body is out of phase with retraction on the other side, resulting in periodic lateral oscillation of the cell body. We present a physical description of keratocyte oscillation in which periodic retraction of the trailing edge is the result of elastic coupling with the leading edge. Consistent with the predictions of this model, the observed frequency of oscillation correlates with cell speed. In addition, decreasing the strength of adhesion to the substrate reduces the elastic force required for retraction, causing cells to oscillate with higher frequency at relatively lower speeds. These results demonstrate that simple elastic coupling between movement at the front of the cell and movement at the rear can generate large-scale mechanical integration of cell behavior.

Balance between cell−substrate adhesion and myosin contraction determines the frequency of motility initiation in fish keratocytes

Significance Symmetry breaking and motility initiation are required for many physiological and pathological processes, but the mechanical mechanisms that drive symmetry breaking are not well understood. Fish keratocytes break symmetry spontaneously, in the absence of external cues, with myosin-driven actin flow preceding rear retraction. Here we combine experimental manipulations and mathematical modeling to show that the critical event for symmetry breaking is a flow-dependent, nonlinear switch in adhesion strength. Moreover, our results suggest that mechanical feedback among actin network flow, myosin, and adhesion is sufficient to amplify stochastic fluctuations in actin flow and trigger symmetry breaking. Our mechanical model for symmetry breaking in the relatively simple keratocyte provides a framework for understanding motility initiation in more complex cell types. 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 forces—contractility and cell−substrate adhesion—determine the likelihood of spontaneous symmetry breaking and motility initiation.

Mechanics of mitochondrial motility in neurons

A properly organized, healthy mitochondrial network is critical for preserving neuronal form and function. Large, elaborately branched neuronal morphologies, energetic demands that fluctuate in time and space, and long neuronal lifespans make the distribution of mitochondria in neurons a particularly complex problem. Moreover, mitochondrial networks are dynamic systems in which mitochondria grow, divide and fuse, move along cytoskeletal filaments, and are degraded in an active fashion. Although the molecular mechanisms that govern mitochondrial motility, in particular, are increasingly well-characterized, the manner in which these mechanisms are coordinated to give rise to the global mitochondrial distribution in neurons is less well understood. Here I review several molecular mechanisms for mitochondrial motility in the context of a general mechanical framework. In this framework, molecular pathways that control mitochondrial movement can be reduced to their effects on the balance of forces that act on mitochondria, driving and opposing movement.

Cell Mechanics at the Rear Act To Steer the Direction of Cell Migration

Motile cells navigate complex environments by changing their direction of travel, generating left-right asymmetries in their mechanical subsystems to physically turn. Currently little is known about how external directional cues are propagated along the length scale of the whole cell and integrated with its force-generating apparatus to steer migration mechanically. We examine the mechanics of spontaneous cell turning in fish epidermal keratocytes and find that the mechanical asymmetries responsible for turning behavior predominate at the rear of the cell, where there is asymmetric centripetal actin flow. Using experimental perturbations we identify two linked feedback loops connecting myosin II contractility, adhesion strength and actin network flow in turning cells that are sufficient to recreate observed cell shapes and trajectories in a computational model. Surprisingly, asymmetries in actin polymerization at the cell leading edge play only a minor role in the mechanics of cell turning – that is, cells steer from the rear. Highlights Fish keratocytes can migrate with persistent angular velocity, straight or in circles. Asymmetry in protrusion at the leading edge is not sufficient to generate persistent turning. Asymmetries in myosin II contraction, actin flow and adhesion at the cell rear cause turns. Our new computational model of migration predicts observed cell trajectories.

Can You Hear Me Now

Auditory communication is central to the social interactions of many animals. In fruit flies, males sing to court females. Coen et al. (2016) demonstrate that males can dynamically adjust the loudness of their songs according to the distance to a female.