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.

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.

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.

Dendrite architecture determines mitochondrial distribution patterns in vivo.

Neuronal morphology influences synaptic connectivity and neuronal signal processing. However, it remains unclear how neuronal shape affects steady-state distributions of organelles like mitochondria. In this work, we investigated the link between mitochondrial transport and dendrite branching patterns by combining mathematical modeling with in vivo measurements of dendrite architecture, mitochondrial motility, and mitochondrial localization patterns in Drosophila HS (horizontal system) neurons. In our model, different forms of morphological and transport scaling rules-which set the relative thicknesses of parent and daughter branches at each junction in the dendritic arbor and link mitochondrial motility to branch thickness-predict dramatically different global mitochondrial localization patterns. We show that HS dendrites obey the specific subset of scaling rules that, in our model, lead to realistic mitochondrial distributions. Moreover, we demonstrate that neuronal activity does not affect mitochondrial transport or localization, indicating that steady-state mitochondrial distributions are hard-wired by the architecture of the neuron.

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.

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.

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 explain the observed cell shapes and trajectories. Notably, 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.

Sequential Nonlinear Filtering of Local Motion Cues by Global Motion Circuits.

Many animals guide their movements using optic flow, the displacement of stationary objects across the retina caused by self-motion. How do animals selectively synthesize a global motion pattern from its local motion components? To what extent does this feature selectivity rely on circuit mechanisms versus dendritic processing? Here we used in vivo calcium imaging to identify pre- and postsynaptic mechanisms for processing local motion signals in global motion detection circuits in Drosophila. Lobula plate tangential cells (LPTCs) detect global motion by pooling input from local motion detectors, T4/T5 neurons. We show that T4/T5 neurons suppress responses to adjacent local motion signals whereas LPTC dendrites selectively amplify spatiotemporal sequences of local motion signals consistent with preferred global patterns. We propose that sequential nonlinear suppression and amplification operations allow optic flow circuitry to simultaneously prevent saturating responses to local signals while creating selectivity for global motion patterns critical to behavior.

Adhesion-Dependent Wave Generation in Crawling Cells.

Dynamic actin networks are excitable. In migrating cells, feedback loops can amplify stochastic fluctuations in actin dynamics, often resulting in traveling waves of protrusion. The precise contributions of various molecular and mechanical interactions to wave generation have been difficult to disentangle, in part due to complex cellular morphodynamics. Here we used a relatively simple cell type-the fish epithelial keratocyte-to define a set of mechanochemical feedback loops underlying actin network excitability and wave generation. Although keratocytes are normally characterized by the persistent protrusion of a broad leading edge, increasing cell-substrate adhesion strength results in waving protrusion of a short leading edge. We show that protrusion waves are due to fluctuations in actin polymerization rates and that overexpression of VASP, an actin anti-capping protein that promotes actin polymerization, switches highly adherent keratocytes from waving to persistent protrusion. Moreover, VASP localizes both to adhesion complexes and to the leading edge. Based on these results, we developed a mathematical model for protrusion waves in which local depletion of VASP from the leading edge by adhesions-along with lateral propagation of protrusion due to the branched architecture of the actin network and negative mechanical feedback from the cell membrane-results in regular protrusion waves. Consistent with our model simulations, we show that VASP localization at the leading edge oscillates, with VASP leading-edge enrichment greatest just prior to protrusion initiation. We propose that the mechanochemical feedbacks underlying wave generation in keratocytes may constitute a general module for establishing excitable actin dynamics in other cellular contexts.

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.

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.

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 cell's 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.

Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis.

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.