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The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells., The arrival of migratory cells at their targets relies on following precise routes within tissues. Here the authors demonstrate that the cell adhesion molecule E-cadherin can control the path of cell migration by confining the site where bleb-type protrusions form within the cell front.

Understanding tissue dynamics and morphogenesis is one of the big challenges of modern biology and has already been studied in detail on the molecular and genetic level during the past decades. The results of these studies led to an integrated view, where the molecular and genetic changes affect force generation and mechanical properties of cells and tissue. However, during the rise of mechanobiology, it turned out that also vice versa, the mechanical properties of the environment largely influence the genetic and molecular composition of many cells. In the context of tissue dynamics and morphogenesis, this single cell–level view needs to be extended to the tissue scale. In this chapter, we will discuss the recent advances in understanding how tissue mechanical changes are the driving forces for different morphogenic collective cell migration during early zebrafish development. We will discuss the first onset of collective migration during zebrafish epiboly, study recent wetting approaches, and review a model that considers viscous effects to explain the doming of the yolk cell. Moreover, we will introduce symmetry-breaking events that are a key element of gastrulation, and finally, we describe how a jamming transition helps in forming the somites of the zebrafish tail.

Nucleus centering in mouse oocytes depends on a gradient of actin-positive vesicle persistence. Modeling coupled to 3D simulations and experimental testing of predictions coming from the simulations demonstrate that this gradient nonspecifically centers large objects during prophase I and meiosis I in oocytes., Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.

Cancer-associated fibroblasts (CAFs) promote cancer cell invasion and dissemination by remodeling the extracellular matrix; however, the mechanism by which CAFs remodel the matrix is still unknown. Attieh et al. show that CAFs induce cancer cell invasion through fibronectin matrix assembly that is mainly mediated by integrin-αvβ3., Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-αvβ3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins αv and β3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-αvβ3 expression as new hallmarks of CAFs that promote tumor invasion.

SUMMARYThe mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, at time scales similar to that of the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not necessary. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement, suggesting that this structure is responsible for redistribution of nesprins towards the front of the nucleus. Two-photon ablation and the use of drugs inhibiting the cytoskeleton demonstrate a pulling force on the nucleus from the front of the cell that is dependent on formin and actomyosin contractility. This elastic recoil is significantly reduced when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant towards the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal—not cancer—stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression., The tumour stroma has altered stiffness and matrix architecture compared to normal tissue, which favours proliferation, and invasion. Here, the authors find that the extracellular matrix produced by normal fibroblasts inhibits cancer cell proliferation through mechanosensitive downregulation of JMJD1a.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells ( Sharpin cpdm ), and mice with a stromal ( S100a4‐Cre ) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro . Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Understanding embryonic development requires the characterization of the forces and the mechanical features that shape cells and tissues within the organism. In addition, experimental application of forces on cells and altering cell and organelle shape allows determining the role such forces play in morphogenesis. Here, we present a holographic optical tweezers-based new microscopic platform for in vivo applications in the context of a developing vertebrate embryo that unlike currently used setups allows simultaneous trapping of multiple objects and rapid comparisons of viscoelastic properties in different locations. This non-invasive technique facilitates a dynamic analysis of mechanical properties of cells and tissues without intervening with embryonic development. We demonstrate the application of this platform for manipulating organelle shape and for characterizing the mechanobiological properties of cells in live zebrafish embryos. The method of holographic optical tweezers as described here is of general interest and can be easily transferred to studying a range of developmental processes in zebrafish, thereby establishing a versatile platform for similar investigations in other organisms. Fluorescent beads injected into zebrafish embryos at 1-cell stage are maintained within the embryos and do not affect their development as observed in the presented 1-day old embryo.

Cells modulate their shape to fulfill specific functions, mediated by the cell cortex, a thin actin shell bound to the plasma membrane. Myosin motor activity, together with actin dynamics, contributes to cortical tension. Here, we examine the individual contributions of actin polymerization and myosin activity to tension increase with a non-invasive method. Cell-sized liposome doublets are covered with either a stabilized actin cortex of preformed actin filaments, or a dynamic branched actin network polymerizing at the membrane. The addition of myosin II minifilaments in both cases triggers a change in doublet shape that is unambiguously related to a tension increase. Preformed actin filaments allow us to evaluate the effect of myosin alone while, with dynamic actin cortices, we examine the synergy of actin polymerization and myosin motors in driving shape changes. Our assay paves the way for a quantification of tension changes triggered by various actin-associated proteins in a cell-sized system.

Neuronal growth is an extremely complex yet reliable process that is directed by a dynamic lamellipodial structure at the tip of every growing neurite, called the growth cone. Lamellipodial edge fluctuations are controlled by the interplay between actin polymerization pushing the edge forward and molecular motor driven retrograde actin flow retracting the actin network. The leading edge switches randomly between extension and retraction processes. We identify switching of "on/off" states in actin polymerization as the main determinant of lamellipodial advancement. Our analysis of motility statistics allows for a prediction of growth direction. This was used in simulations explaining the amazing signal detection capabilities of neuronal growth by the experimentally found biased stochastic processes. Our measurements show that the intensity of stochastic fluctuations depend on changes in the underlying active intracellular processes and we find a power law eta = a*x(alpha) with exponent alpha = 2.63 +/- 0.12 between noise intensity eta and growth cone activity x, defined as the sum of protrusion and retraction velocity. Differences in the lamellipodial dynamics between primary neurons and a neuronal cell line further suggests that active processes tune the observed stochastic fluctuations. This hints at a possible role of noise intensity in determining signal detection sensitivity.

To keep the mechanical integrity of an organism it seems obvious that cells, as the building blocks, must be solid. Although it is clear that switching to a fluid would be catastrophic for organization of the body, it turns out that living cells do change their mechanical properties to a more fluid-like behavior when it comes to migration and force generation. Being fluid-like allows cells to adapt to any arbitrary shape posed by the environment, which is crucial for movement through complex tissue. The mechanical integrity of healthy cells is therefore closely regulated to ensure that cells are solid enough to maintain tissue shape while also being fluid enough to allow dynamic remodeling. Physics provides powerful tools in the framework of viscoelasticity to characterize this fundamental solid and fluid-like behavior (1), and it is evident that cells need to dynamically regulate their viscoelastic properties to support physiological pressures and forces generated during lung expansion, muscle contraction, blood filtration, etc., while still allowing growth, remodeling, and repair over the lifetime of the organism. However, when this precise mechanical regulation is disturbed, cells often transition to diseased states (2). In PNAS, Ehrlicher et al. (3) study a genetic defect in the actin cross-linker alpha-actinin 4 that is known to lead to the severe kidney disease focal segmental glomerulosclerosis. Their study shows that the mutation affects cell movement, force generation, and cytoplasmic mobility, thus providing a connection between physical properties at the molecular scale and human disease.

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Many different cell types including fibroblasts, smooth muscle cells, endothelial cells, and cancer cells exert traction forces on the fibrous components of the extracellular matrix. This can be observed as matrix contraction both macro- and microscopically in three-dimensional (3D) tissues models such as collagen type I gels. The quantification of local contraction at the micron scale, including its directionality and speed, in correlation with other parameters such as cell invasion, local protein or gene expression, can provide useful information to study wound healing, organism development, and cancer metastasis. In this article, we present a set of tools to quantify the flow dynamics of collagen contraction, induced by cells migrating out of a multicellular cancer spheroid into a three-dimensional (3D) collagen matrix. We adapted a pseudo-speckle technique that can be applied to bright-field and fluorescent microscopy time series. The image analysis presented here is based on an in-house written software developed in the Matlab (Mathworks) programming environment. The analysis program is freely available from GitHub following the link: http://dx.doi.org/10.5281/zenodo.10116. This tool provides an automatized technique to measure collagen contraction that can be utilized in different 3D cellular systems.

Cell motility and division are complex mechanisms that involve drastic cell shape changes. A crucial role is played by the actin cortex, which, together with the molecular motor myosin, regulates the production of cellular tension (1). A better understanding of the interplay between actin network dynamics and myosin-driven contractility is necessary to elucidate the detailed mechanism of contraction in a dynamic, out of equilibrium, constantly polymerizing and depolymerizing actin structure. In order to dissect this dual actin-myosin contribution we make advantage of biomimetic systems: cell-sized liposomes indeed offer the possibility to retain only the essential players involved in contraction mechanisms and to finely control the physical and biochemical conditions to mimic cell-like shape changes (2).Particularly we study acto-myosin contraction mechanisms on cell-sized doublets, which enable overcoming the spherical symmetry of single liposomes: with this geometry, shape changes, and hence tension build-up, can be fully characterised by recording contact angle variations. A polymerizing actin network is built at the outer surface of doublets. A fluorescently labelled actin is used and simultaneous detection of phase contrast and fluorescence images allow following actin cortex dynamics and further shape changes induced by the addition of myosin motors (3). A micropipette aspiration technique is also adopted to further characterize these cell-like shape changes (4).This reconstituted doublet system allows us to decouple biochemical and physical aspects of actin network dynamics and fully dissect the dual role of actin and myosin contractility, thus significantly improving our understanding of acto-myosin driven cell shape changes.1.Blanchoin L et al. Physiol Rev 2014, 94:235-263.2.Murrell M et al. Nat Rev Mol Cell Biol. 2015, 16(8):486-98.3.Carvalho K et al. Proc Natl Acad Sci U S A. 2013, 110(41):16456-16461.4.Campillo C et al. Biophys J 2013, 104:1248-1256.

Cell migration is essential for morphogenesis, for organ formation and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model to study this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility and with the ability of the cells to reach their target.

SummaryMigrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments [1–5]. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth [6–9]. However, tension must be tightly controlled, because if released, FAs disassemble [3, 10–12]. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion [9, 12]. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. Here, we show that the actin-bundling protein fascin crosslinks the actin filaments into parallel bundles at the stress fibers’ termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. We also show that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs.

Summary Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors., Graphical Abstract, Highlights • CIL in embryonic dispersing hemocytes requires choreographed changes in motility • Tracking actin flow in vivo reveals synchronous changes in colliding actin networks • An inter-cellular actin-clutch between colliding cells induces lamellar tension • Tension buildup and release orchestrate the CIL response, Contact inhibition of locomotion in Drosophila macrophages involves mechanical coupling of colliding actin networks. The resultant buildup in lamellar tension orchestrates the response and choreographs cell repulsion.

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

International audience; Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an 'outside geometry'. The actin shell is either grown from an activator of actin polymeriz-ation immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.

We present a novel technique to noninvasively control the growth and turning behavior of an extending neurite. A highly focused infrared laser, positioned at the leading edge of a neurite, has been found to induce extension/turning toward the beam's center. This technique has been used successfully to guide NG108–15 and PC12 cell lines [Ehrlicher, A., Betz, T., Stuhrmann, B., Koch, D. Milner, V. Raizen, M. G., and Kas, J. (2002). Guiding neuronal growth with light. Proc. Natl. Acad. Sci. USA 99, 16024–16028], as well as primary rat and mouse cortical neurons [Stuhrmann, B., Goegler, M., Betz, T., Ehrlicher, A., Koch, D., and Kas, J. (2005). Automated tracking and laser micromanipulation of cells. Rev. Sci. Instr . 76, 035105]. Optical guidance may eventually be used alone or with other methods for controlling neurite extension in both research and clinical applications.

Understanding and controlling neuronal growth are basic objectives in neuroscience, biology, biophysics, and biomedicine, and are vital for the formation of neural circuits in vitro, as well as for nerve regeneration in vivo. All molecular stimuli for neuronal growth eventually address the polymeric cytoskeleton, which advances a neurite's leading edge also known as the growth cone. We have shown that optical forces of a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization-driven intracellular machinery. In actively extending growth cones, a laser spot placed at specific areas of the neurite's leading edge affects the growth speed, the direction taken by a growth cone, and the splitting of a growth cone. This novel optical tool manipulates a natural biological process, the cytoskeleton driven morphological changes in growth cones, with potential applications in the formation of neuronal networks and in understanding growth cone motility. The current apparatus combines optical tweezers, phase contrast and fluorescence imaging, and real-time shape detection. Automated and dynamically readjusted irradiation of the growth cone is used to examine and to influence structural and morphological changes of neuronal growth.

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro , as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457–10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156–159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517–1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.

The motility of cells is a multifaceted and complicated cytoskeletal process. Significant inroads can be made into gaining a more detailed understanding, however, by focusing on the smaller, more simple subunits of the motile system in an effort to isolate the essential protein components necessary to perform a certain task. Identification of such functional modules has proven to be an effective means of working towards a comprehensive understanding of complex, interacting systems. By following a bottom-up approach in studying minimal actin-related sub-systems for keratocyte motility, we revealed several fundamentally important effects ranging from an estimation of the force generated by the polymerization of a single actin filament, to assembly dynamics and the production of force and tension of composite actin networks, to the contraction of actin networks or smaller bundled structures by the motor myosin II. While even motile keratocyte fragments represent a far more complex situation than the simple reconstituted systems presented here, clear parallels can be seen between in vivo cell motility and the idealized in vitro functional modules presented here, giving more weight to their continued focus.