Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base.

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvβ6 and α9β1 engagement, rather than α5β1. Moreover, αvβ6-driven migration occurs independently of latent TGF-β activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour–stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments. INTRODUCTIONCrawling cell motion involves a cycle of several distinct processes: protrusion at the front of the cell, attachment to the substratum, and forward translocation of the cell body followed or accompanied by detachment and withdrawal of the rear of the cell. Crawling motion is thought to be dependent on the actin-myosin II cytoskeletal system (Mitchison and Cramer, 1996): protrusion is thought to be driven by the assembly of actin network, which is anchored to the substratum through integrin-containing adhesions; forward translocation of the cell body and contraction of the rear are thought to depend on the interaction of the actin network with the motor protein myosin II. Actin assembly during the protrusion at the leading edge of motile cells has recently received the most attention both in experimental (Pantaloni et al., 2001;Pollard and Borisy, 2003;Ridley et al., 2003) and theoretical studies (for review, see Mogilner, 2006). In contrast, the mechanisms involved in the forward translocation of the cell body remain largely unclear: the exact layout and the mode of action of the contractile actin-myosin II machinery are controversial. Qualitative models proposed in the literature include shortening of small contractile units similar to muscle sarcomeres, myosin II-dependent transport along uniformly polarized actin arrays, and a dynamic network contraction mechanism where contraction results from alignment of actin filaments by myosin II assemblies (Cramer, 1999;Verkhovsky et al., 1999a). More sophisticated biophysical models aim to understand the contractile cytoskeletal machinery in quantitative physical terms. In the past, considering the cytoskeleton as a gel made of cross-linked semiflexible polymers has helped understanding its passive visco-elastic properties. More recently, attempts were made to develop biophysical models taking into account intrinsic activity of the cytoskeleton: polarized assembly of acti...

Correspondence sandoz@unice.fr In BriefRoyal et al. demonstrate that migraineassociated frameshift mutations of TRESK, a two-pore-domain K+ channel, lead to the production of a second protein fragment, which carries the pathophysiological function by inhibiting TREK1 and 2, due to a mechanism called frameshift mutation-induced alternative translation initiation (fsATI).

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM.Analyses revealed that therapy resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and forcemediated ECM remodeling through activation of actin-dependent mechanosensors Yesassociated protein (YAP) and Myocardin-related transcription factor (MRTF). Shortterm inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP and MRTFdependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacological targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCEThese findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing and therapy resistance.

Neoformation of intercellular adherens junctions accompanies the differentiation of fibroblasts into contractile myofibroblasts, a key event during development of fibrosis and in wound healing. We have previously shown that intercellular mechanical coupling of stress fibres via adherens junctions improves contraction of collagen gels by myofibroblasts. By assessing spontaneous intracellular Ca2+ oscillations, we here test whether adherens junctions mechanically coordinate myofibroblast activities. Periodic Ca2+ oscillations are synchronised between physically contacting myofibroblasts and become desynchronised upon dissociation of adherens junctions with function-blocking peptides. Similar uncoupling is obtained by inhibiting myofibroblast contraction using myosin inhibitors and by blocking mechanosensitive ion channels using Gd3+ and GSMTx4. By contrast, gap junction uncouplers do not affect myofibroblast coordination. We propose the following model of mechanical coupling for myofibroblasts: individual cell contraction is transmitted via adherens junctions and leads to the opening of mechanosensitive ion channels in adjacent cells. The resulting Ca2+ influx induces a contraction that can feed back on the first cell and/or stimulate other contacting cells. This mechanism could improve the remodelling of cell-dense tissue by coordinating the activity of myofibroblasts.

A gradient of PI(4,5)P2 formed by phospholipid synthesis, diffusion, and regulated turnover is crucial for filamentous growth.

Lamellipodia of crawling cells represent both the motor for cell advance and the primary building site for the actin cytoskeleton. The organization of actin in the lamellipodium reflects actin dynamics and is of critical importance for the mechanism of cell motility. In previous structural studies, the lamellipodial actin network was analyzed primarily by electron microscopy (EM). An understanding of lamellipodial organization would benefit significantly if the EM data were complemented and put into a kinetic context by establishing correspondence with structural features observable at the light microscopic level in living cells. Here, we use an enhanced phase contrast microscopy technique to visualize an apparent long-range diagonal actin meshwork in the advancing lamellipodia of living cells. Visualization of this meshwork permitted a correlative light and electron microscopic approach that validated the underlying organization of lamellipodia. The linear features in the light microscopic meshwork corresponded to regions of greater actin filament density. Orientation of features was analyzed quantitatively and compared with the orientation of actin filaments at the EM level. We infer that the light microscopic meshwork reflects the orientational order of actin filaments which, in turn, is related to their branching angle. INTRODUCTIONProtrusion at the leading edge of most crawling cells is believed to be driven by assembly of actin filaments (Mogilner and Oster, 1996;Borisy and Svitkina, 2000;Pollard et al., 2000;Pantaloni et al., 2001). One of the major protrusive specializations of the cell edge is the lamellipodium, a flat, sheet-like region of the cell filled with a dense network of actin. The dynamics of actin assembly in lamellipodia and the nature of the molecular events that are orchestrated to produce protrusion represent central problems in the cell motility field. Conclusions about actin dynamics have been inferred from structural studies under the assumption that the dynamics of the actin network are imprinted in its structure. Given that the dimensions of actin filaments themselves as well as the average interfilament distances in the cell are well below the resolution of light microscopy, electron microscopy (EM) has long been the primary tool for analyzing the structure of lamellipodia.Important structural features established by EM are the uniform polarity of actin filaments, the preferred end for elongation (barbed end) pointing toward the leading edge of the cell (Small et al., 1978;Svitkina et al., 1997), and the dendritic organization of the actin network (Svitkina et al., 1997) with the Arp 2/3 complex present at the branching points of the filaments (Svitkina et al., 1999). These findings in combination with biochemical data and functional analysis (Machesky and Insall, 1998;Loisel et al., 1999;Mullins and Pollard, 1999) led to the formulation of a conceptual framework for actin dynamics featuring dendritic nucleation of filaments mediated by the Arp 2/3 complex and rapid turnover of the ...