Kinneret Keren

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.

The Shape of Motile Cells

Motile cells - fan-like keratocytes, hand-shaped nerve growth cones, polygonal fibroblasts, to name but a few - come in different shapes and sizes. We discuss the origins of this diversity as well as what shape tells us about the physics and biochemistry underlying cell movement. We start with geometric rules describing cell-edge kinetics that govern cell shape, followed by a discussion of the underlying biophysics; we consider actin treadmilling, actin-myosin contraction, cell-membrane deformations, adhesion, and the complex interactions between these modules, as well as their regulation by microtubules and Rho GTPases. Focusing on several different cell types, including keratocytes and fibroblasts, we discuss how dynamic cell morphology emerges from the interplay between the different motility modules and the environment.

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.

Intracellular fluid flow in rapidly moving cells

Cytosolic fluid dynamics have been implicated in cell motility because of the hydrodynamic forces they induce and because of their influence on transport of components of the actin machinery to the leading edge. To investigate the existence and the direction of fluid flow in rapidly moving cells, we introduced inert quantum dots into the lamellipodia of fish epithelial keratocytes and analysed their distribution and motion. Our results indicate that fluid flow is directed from the cell body towards the leading edge in the cell frame of reference, at about 40% of cell speed. We propose that this forward-directed flow is driven by increased hydrostatic pressure generated at the rear of the cell by myosin contraction, and show that inhibition of myosin II activity by blebbistatin reverses the direction of fluid flow and leads to a decrease in keratocyte speed. We present a physical model for fluid pressure and flow in moving cells that quantitatively accounts for our experimental data.

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.

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.

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.

From DNA to transistors

The rapid advance in molecular biology and nanotechnology opens up the possibility to explore the interface between biology and electronics at the single-molecule level. We focus on the organization of molecular electronic circuits. Interconnecting an immense number of molecular devices into a functional circuit and constructing a framework for integrated molecular electronics requires new concepts. A promising avenue relies on bottom-up assembly where the information for the circuit connectivity and functionality is embedded in the molecular building blocks. Biology can provide concepts and mechanisms for advancing this approach, but there is no straightforward way to apply them to electronics since biological molecules are essentially electrically insulating. Bridging the chasm between biology and electronics therefore presents great challenges. Circuit organization on the molecular scale is considered and contrasted with the levels of organization presented by the living world. The discussion then focuses on our proposal to harness DNA and molecular biology to construct the scaffold for integrated molecular electronics. DNA metallization is used to convert the DNA scaffold into a conductive one. We present the framework of sequence-specific molecular lithography based on the biological mechanism of homologous genetic recombination and carried out by the bacterial protein RecA. Molecular lithography enables us to use the information encoded in the scaffold DNA molecules for directing the construction of an electronic circuit. We show that it can lead all the way from DNA molecules to working transistors in a test-tube. Carbon nanotubes are incorporated as the active electronic components in the DNA-templated transistors. Our approach can, in principle, be applied to the fabrication of larger-scale electronic circuits. The realization of complex DNA-based circuits will, however, require new concepts and additional biological machinery allowing, for example, feedback from the electronic functionality to direct the assembly process and adaptation mechanisms.

Symmetry breaking in reconstituted actin cortices

The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide. DOI: http://dx.doi.org/10.7554/eLife.01433.001

Segmentation and tracking of live cells in phase-contrast images using directional gradient vector flow for snakes.

Cell shape is an important characteristic of the physiological state of a cell and is used as a primary read‐out of cell behaviour in various assays. Automated accurate segmentation of cells in microscopy images is hence of large practical importance in cell biology. We report a simple algorithm for automated cell segmentation in high‐magnification phase‐contrast images, which takes advantage of the characteristic directionality of the local image intensity gradient at cellular boundaries due to the ‘halo‐effect’. We employ a two‐step algorithm in which a gradient vector flow (GVF) field is first used to direct active contours to an approximate cell boundary. A directional GVF (DGVF) field is then calculated by considering only edges for which the image intensity gradient is directed outwards with respect to the approximate cell contour. Subsequently, the DGVF field is used to refine the cell contour, by directing active contours to edges with the desired gradient directionality. This method allows us to accurately segment cells in an image series, as well as follow the dynamics of cell shape over time in an automated fashion.