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Cell growth depends upon formation of cell-matrix adhesions, but mechanisms detailing the transmission of signals from adhesions to control proliferation are still lacking. Here, we find that the scaffold protein talin undergoes force-induced cleavage in early adhesions to produce the talin rod fragment that is needed for cell cycle progression. Expression of noncleavable talin blocks cell growth, adhesion maturation, proper mechanosensing, and the related property of EGF activation of motility. Further, the expression of talin rod in the presence of noncleavable full-length talin rescues cell growth and other functions. The cleavage of talin is found in early adhesions where there is also rapid turnover of talin that depends upon calpain and TRPM4 activity as well as the generation of force on talin. Thus, we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions.

The role of myristoylation in the localization and catalytic activity of Src at focal adhesions was investigated by live-cell imaging and site-directed mutagenesis. Although the majority of activated Src molecules are localized at focal adhesions, it is unclear how activated Src molecules are recruited to focal adhesions. Because Src is activated at the cell membrane, translocation of Src to cell membranes is considered to be essential for its recruitment to focal adhesions. Membrane-targeting-deficient Src mutant SrcG2A localizes at focal adhesions, indicating direct recruitment of Src from cytosol to focal adhesions. Furthermore, directly recruited Src molecules are shown to enhance paxillin dynamics at focal adhesions. These results reveal that the regulation of Src activation and translocation is more complex than previously suggested.

Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK)-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0418-y) contains supplementary material, which is available to authorized users.

Cell-matrix adhesion complexes are multi-protein structures linking the extracellular matrix (ECM) to the cytoskeleton. They are essential to both cell motility and function by bidirectionally sensing and transmitting mechanical and biochemical stimulations. Several types of cell-matrix adhesions have been identified and they share many key molecular components, such as integrins and actin-integrin linkers. Mechanochemical coupling between ECM molecules and the actin cytoskeleton has been observed from the single cell to the single molecule level and from immune cells to neuronal cells. However, the mechanisms underlying force regulation of integrin-mediated mechanotransduction still need to be elucidated. In this review article, we focus on integrin-mediated adhesions and discuss force regulation of cell-matrix adhesions and key adaptor molecules, three different force-dependent behaviors, and molecular mechanisms for mechanochemical coupling in force regulation.

Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7’s NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement., The spectraplakin protein ACF7 binds to actin at focal adhesions and targets microtubule plus ends to focal adhesions, promoting their disassembly. Here the authors reveal that ACF7 is phosphorylated by Src/FAK, and this regulates actin binding and focal adhesion dynamics in vitro and in vivo.

Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Cas in vitro in a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration., Summary: Tensin 1 links phosphorylated Cas to actin inward flux, facilitating the force transmission from the motile system (i.e. actomyosin contraction) to the stationary parts (i.e. adhesion complexes) in migrating cells.

Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or “clutch” myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show inXenopusspinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuatedin vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues.SIGNIFICANCE STATEMENTHere, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidancein vivo. Consistent with this notion, we found that RF rates of spinal neuron growth cones were slowerin vivothan what was observedin vitro. Together, our study provides the best evidence that growth cone–ECM adhesions clutch RF locally to guide axonsin vivo.

Sun, Guo, and Fässler review the function and regulation of integrin-mediated mechanotransduction and discuss how its dysregulation impacts cancer progession., Cells can detect and react to the biophysical properties of the extracellular environment through integrin-based adhesion sites and adapt to the extracellular milieu in a process called mechanotransduction. At these adhesion sites, integrins connect the extracellular matrix (ECM) with the F-actin cytoskeleton and transduce mechanical forces generated by the actin retrograde flow and myosin II to the ECM through mechanosensitive focal adhesion proteins that are collectively termed the “molecular clutch.” The transmission of forces across integrin-based adhesions establishes a mechanical reciprocity between the viscoelasticity of the ECM and the cellular tension. During mechanotransduction, force allosterically alters the functions of mechanosensitive proteins within adhesions to elicit biochemical signals that regulate both rapid responses in cellular mechanics and long-term changes in gene expression. Integrin-mediated mechanotransduction plays important roles in development and tissue homeostasis, and its dysregulation is often associated with diseases.

SummaryIntegrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth.

The shootin1–cortactin interaction participates in netrin-1–induced F-actin–adhesion coupling and in the promotion of traction forces for axon outgrowth., Motile cells transduce environmental chemical signals into mechanical forces to achieve properly controlled migration. This signal–force transduction is thought to require regulated mechanical coupling between actin filaments (F-actins), which undergo retrograde flow at the cellular leading edge, and cell adhesions via linker “clutch” molecules. However, the molecular machinery mediating this regulatory coupling remains unclear. Here we show that the F-actin binding molecule cortactin directly interacts with a clutch molecule, shootin1, in axonal growth cones, thereby mediating the linkage between F-actin retrograde flow and cell adhesions through L1-CAM. Shootin1–cortactin interaction was enhanced by shootin1 phosphorylation by Pak1, which is activated by the axonal chemoattractant netrin-1. We provide evidence that shootin1–cortactin interaction participates in netrin-1–induced F-actin adhesion coupling and in the promotion of traction forces for axon outgrowth. Under cell signaling, this regulatory F-actin adhesion coupling in growth cones cooperates with actin polymerization for efficient cellular motility.

Focal adhesions (FAs) link the extracellular matrix (ECM) to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signaling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FA is unknown. Using interferometric photo-activation localization (iPALM) super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FA. Inactive vinculin localizes to the lower integrin signaling layer in FA by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FA where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FA at the nano-scale to regulate vinculin activation and function.

The tripartite motif containing (TRIM) proteins are a large family of proteins that have been implicated in many biological processes including cell differentiation, apoptosis, transcriptional regulation, and signaling pathways. Here, we show that TRIM15 co-localized to focal adhesions through homo-dimerization and significantly suppressed cell migration. Domain mapping analysis indicated that B-box2 and PRY domains were essential for TRIM15 localization to focal adhesions and inhibition of cell migration. Our protein-protein interaction screen of TRIM15 with the integrin adhesome identified several TRIM15 interacting proteins including coronin 1B, cortactin, filamin binding LIM protein1, and vasodilator-stimulated phosphoprotein, which are involved in actin cytoskeleton dynamics. TRIM15 expression was tissue-restricted and downregulated in colon cancer. Level of TRIM15 expression was associated with colon cancer cell migration, as well as both in vitro and in vivo tumor growth. These data provide novel insights into the role of TRIM15 as an additional component of the integrin adhesome, regulating cell migration, and suggest that TRIM15 may function as a tumor suppressor of colon cancer.

The LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes., The linker of nucleoskeleton and cytoskeleton (LINC) complex allows cells to actively control nuclear position by coupling the nucleus to the cytoplasmic cytoskeleton. Nuclear position responds to the formation of intercellular adhesions through coordination with the cytoskeleton, but it is not known whether this response impacts adhesion function. In this paper, we demonstrate that the LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes. Mice deficient for Sun2 exhibited irregular hair follicle intercellular adhesions, defective follicle structure, and alopecia. Primary mouse keratinocytes lacking Sun2 displayed aberrant nuclear position in response to adhesion formation, altered desmosome distribution, and mechanically defective adhesions. This dysfunction appeared rooted in a failure of Sun2-null cells to reorganize their microtubule network to support coordinated intercellular adhesion. Together, these results suggest that cross talk between the nucleus, cytoskeleton, and intercellular adhesions is important for epidermal tissue integrity.

Developing tissues change shape, and tumors initiate spreading, through collective cell motility. Conserved mechanisms by which tissues initiate motility into their surroundings are not known. We investigated cytoskeletal regulators during collective invasion by mouse tumor organoids and epithelial MDCK acini undergoing branching morphogenesis. Inhibition of formins, but not Arp2/3, prevented the formation of migrating cell fronts in both cell types. MDCK cells depleted of the formin protein Dia1 formed polarized acini and could execute planar cell motility, either within the acinus or in 2D scattering assays. However, Dia1 was required to form protrusions into the collagen matrix. Live imaging of actin, myosin, and collagen in control acini revealed adhesions that deformed individual collagen fibrils, while Dia1-depleted acini exhibited unstable adhesions with minimal collagen deformation. This work identifies Dia1-mediated adhesions as essential regulators of tissue shape changes, through their role in focal adhesion maturation.

α6β4 integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of β4 integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α6 integrin retains its punctate distribution. Moreover, in β4 integrin knockdown cells, we observe a loss of β1 integrin from focal adhesions and an enhanced association with α6 integrin. We confirmed the switch in the β integrin binding partner of α6 integrin in the knockdown cells by immunoprecipitation. We next investigated the role of β4 integrin in collective cell migration. Wounded monolayers of β4 integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of β4 integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of β4 integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which β4 integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from β4 integrin deficiency. Together, our results support the hypothesis that α6β4 integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.

Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described ‘spatial rheostat’ controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs. DOI: http://dx.doi.org/10.7554/eLife.23172.001, eLife digest In animal bodies, a mesh of proteins – known the extracellular matrix – holds cells together to give the body shape and make it more stable. Cells bind to the matrix using structures called focal adhesions. However, cells do not always stay in one place: in young animals, certain cells need to move around the body to reach their final destination. Adult animals also have some cells that are able to move, for example, to close wounds. The cells move when the focal adhesions that hold these cells in place are taken apart and then rebuilt. These processes are very dynamic and happen all the time when cells move. They are normally tightly controlled to ensure that cells only migrate under appropriate conditions. However, focal adhesions are less well regulated in cancer cells, allowing the cells to migrate away from a tumour to form new tumours elsewhere in the body. Focal adhesions are large structures that contain many proteins. These proteins include FAK and Src, which are particularly important and have been well studied. In order to better understand how focal adhesions are taken apart, Schoenherr et al. wanted to discover new proteins that interact with FAK in skin cancer cells from mice. The experiments show that FAK binds to a protein called Ambra1, which is known to control how other proteins move around inside cells. Ambra1 and FAK work together to regulate the movement of Src away from focal adhesions and into the cell. Furthermore, Ambra1 belongs to a larger network of proteins within the cancer cells that regulates the location of Src. By changing the levels of Src and FAK at focal adhesions, Ambra1 and its other binding partners can control whether the cancer cells are attached to the extracellular matrix or are free to migrate. Overall this work shows that the location and activity of Src within cells needs to be carefully controlled to stop the cells from moving at the wrong time. Further experiments will aim to understand which other proteins are involved in this network and how they contribute to the growth of cancer cells and the spread of tumours around the body. DOI: http://dx.doi.org/10.7554/eLife.23172.002

During tissue morphogenesis, cellular rearrangements give rise to a large variety of three-dimensional structures. Final tissue architecture varies greatly across organs, and many develop to include combinations of folds, tubes, and branched networks. To achieve these different tissue geometries, constituent cells must follow different programs that dictate changes in shape and/or migratory behavior. One essential component of these changes is the remodeling of cell-cell adhesions. Invasive migratory behavior and separation between tissues require localized breakdown of cadherin-mediated adhesions. Conversely, tissue folding and fusion require the formation and reinforcement of cell-cell adhesions. Cell-cell adhesion plays a critical role in tissue morphogenesis; its manipulation may therefore prove to be invaluable in generating complex topologies ex vivo. Recapitulating these shapes in engineered tissues would enable a better understanding of how these processes occur in vivo, and may lead to improved design of organs for clinical applications. In this review, we discuss work investigating the formation of folds, tubes, and branched networks with an emphasis on known or possible roles for cell-cell adhesion. We then examine recently developed tools that could be adapted to manipulate cell-cell adhesion in engineered tissues.

Interactions between integrin-mediated adhesions and the extracellular matrix (ECM) are important regulators of cell migration and spreading. However, mechanisms by which extracellular ligands regulate cell migration and spreading in response to changes in substratum concentration are not well understood. Semaphorin 3A (Sema3A) has been shown to inhibit cell motility and alter integrin signaling in various cell types. We propose that Sema3A alters focal adhesions to modulate breast carcinoma cell migration and spreading on substrata coated with different concentrations of ECM. We demonstrate that Sema3A inhibits MDA-MB-231 cell migration and spreading on substrata coated with high concentrations of collagen and fibronectin but enhances migration and spreading at lower concentrations of collagen and fibronectin. Sema3A increases focal adhesion kinase phosphorylation at tyrosine 397 (pFAK397) at focal adhesions on all substratum concentrations of collagen and fibronectin but decreased pFAK397levels on laminin. Rho-associated protein kinase (ROCK) inhibition blocks the Sema3A-mediated effects on cell migration, spreading, and pFAK397at focal adhesions when cultured on all concentrations of collagen. These results suggest that Sema3A shifts the optimal level of cell-matrix adhesions to a nonoptimal ECM coating concentration, in particular collagen, to yield maximal cell migration and spreading that may be mediated through a ROCK-dependent mechanism.

Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration. NHMRC Grant 632515

Material features proved to exert a potent influence on cell behaviour in terms of adhesion, migration and differentiation. In particular, biophysical and biochemical signals on material surfaces are able to affect focal adhesion distribution and cytoskeletal assemblies, which are known to regulate signalling pathways that ultimately influence cell fate and functions. However, a general, unifying model that correlates cytoskeletal-generated forces with genetic events has yet to be developed. Therefore, it is crucial to gain a better insight into the material-cytoskeleton crosstalk in order to design and fabricate biomaterials able to govern cell fate more accurately. In this work, we demonstrate that confining focal adhesion distribution and growth dramatically alters the cytoskeleton's structures and dynamics, which in turn dictate cellular and nuclear shape and polarization. MC3T3 preosteoblasts were cultivated on nanograted polydimethylsiloxane substrates and a thorough quantification – in static and dynamic modes – of the morphological and structural features of focal adhesions and cytoskeleton was performed. Nanoengineered surfaces provided well-defined zones for focal adhesions to form and grow. Unique cytoskeletal structures spontaneously assembled when focal adhesions were confined and, in fact, they proved to be very effective in deforming the nuclei. The results here presented provide elements to engineer surfaces apt to guide and control cell behaviour through the material-cytoskeleton-nucleus axis.

Summary Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force., Graphical Abstract, Highlights • Nontransformed fibroblasts on RGD membranes form podosome-like protrusions • Nanopatterned RGD membranes enable traction force, suppressing protrusion formation • Local activation of PI3K transforms prepodosomal-like RGD clusters • PIP3-bound RhoA GAP ARAP3 is recruited at the protrusion and downregulates RhoA-GTP, Outside-in integrin activation plays a critical role in cell adhesion and migration. Yu, Sheetz, and colleagues now report that plating cells onto fluid RGD membranes, thus inhibiting the development of traction force, causes the formation of podosome-like protrusions. The podosome-like adhesions on RGD membranes lack matrix metalloproteinases but share many other molecular components with classic podosomes in macrophages. The authors demonstrate that podosome-like protrusion formation is mediated by a PIP3-dependent pathway and is further aided by ARAP3-mediated RhoA inactivation.

Focal adhesions are large protein complexes organized at the basal surface of cells, which physically connect the extracellular matrix to the cytoskeleton and have long been speculated to mediate cell migration. However, whether clustering of these molecular components into focal adhesions is actually required for these proteins to regulate cell motility is unclear. Here we use quantitative microscopy to characterize descriptors of focal adhesion and cell motility for mouse embryonic fibroblasts and human fibrosarcoma cells, across a wide range of matrix compliance and following genetic manipulations of focal adhesion proteins (vinculin, talin, zyxin, FAK, and paxilin). This analysis reveals a tight, biphasic gaussian relationship between mean size of focal adhesions (not their number, surface density, or shape) and cell speed. The predictive power of this relationship is comprehensively validated by disrupting nonfocal adhesion proteins (α-actinin, F-actin, and myosin II) and subcellular organelles (mitochondria, nuclear DNA, etc.) not known to affect either focal adhesions or cell migration. This study suggests that the mean size of focal adhesions robustly and precisely predicts cell speed independently of focal adhesion surface density and molecular composition.

Talin-mediated integrin activation drives integrin-based adhesions. A simple binary switch—vinculin competitively displacing RIAM from talin—is found to play a central role in the maturation and evolving functions of integrin-based adhesions., Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin–integrin interactions—vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)—in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.

Living cells are continuously exposed to mechanical cues, and can translate these signals into biochemical information (e.g. mechanotransduction). This process is crucial in many normal cellular functions, e.g. cell adhesion, migration, proliferation, and survival, as well as the progression of diseases such as cancer. Focal adhesions are the major sites of interactions between extracellular mechanical environments and intracellular biochemical signalling molecules/cytoskeleton, and hence focal adhesion proteins have been suggested to play important roles in mechanotransduction. Here, we overview the current molecular understanding in mechanotransduction occurring at focal adhesions. We also introduce recent studies on how extracellular matrix and mechanical microenvironments contribute to the development of cancer.

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. Here we show that suppressor of cytokine signaling 6 (SOCS6) and Cullin 5, two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or if cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration. DOI: http://dx.doi.org/10.7554/eLife.17440.001, eLife digest Animal cells can move in the body, for example to heal a wound, by protruding a leading edge forwards, attaching it to the surroundings and then pulling against these new attachments while disassembling the older ones. Mechanical forces regulate the assembly and disassembly of these attachments, known as focal adhesions, and so do signals from outside the cell that are transmitted to the adhesions via specialized proteins. However, it was not clear how the assembly and disassembly of adhesions is coordinated. CRL5 is a ubiquitin ligase, an enzyme that can mark other proteins for destruction. Cells migrate more quickly if CRL5 is inhibited, and so Teckchandani and Cooper set out to uncover whether CRL5 affects the assembly and disassembly of focal adhesions. The experiments showed that human cells lacking a crucial component of the CRL5 complex, SOCS6, disassemble adhesions faster than normal cells, but only at their leading edge and not at the rear. Teckchandani and Cooper also found that SOCS6 localizes to the leading edge by binding to a focal adhesion protein called Cas. Shortly after the attachments assemble, the Cas protein becomes tagged with a phosphate group and then acts to promote the adhesion to disassemble. Further experiments indicated that Cas was marked by the CRL5 complex and possibly destroyed while in or very close to the leading edge adhesions, slowing their disassembly. Together, these findings suggest that by binding Cas, SOCS6 regulates the turnover of adhesions, specifically by inhibiting disassembly and allowing adhesions to grow at the leading edge. Since SOCS6 is not present in adhesions outside of the leading edge, this may help explain how the older adhesions are disassembled. Future studies could next focus on the exact sequence of events that occur in focal adhesions after the CRL5 complex binds to Cas as the cell migrates. DOI: http://dx.doi.org/10.7554/eLife.17440.002

Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs via its N-terminus, requiring both its Cdc42/Rac Interactive Binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001, eLife digest Animal cells are organized into tissues and organs. A scaffold-like framework outside of the cells called the extracellular matrix provides support to the cells and helps to hold them in place. Cells attach to the extracellular matrix via structures called focal adhesions on the cell surface; these structures contain a protein called talin. For a cell to be able to move, the existing focal adhesions must be broken down and new adhesions allowed to form. This process is regulated by the delivery and removal of different materials along fibers called microtubules. Microtubules can usually grow and shrink rapidly, but near focal adhesions they are captured at the surface of the cell and become more stable. However, it is not clear how focal adhesions promote microtubule capture and stability. Bouchet et al. found that a protein called KANK1 binds to the focal adhesion protein talin in human cells grown in a culture dish. This allows KANK1 to recruit microtubules to the cell surface around the focal adhesions by binding to particular proteins that are associated with microtubules. Disrupting the interaction between KANK1 and talin by making small alterations in these two proteins blocked the ability of focal adhesions to capture surrounding microtubules. The next step following on from this work will be to find out whether this process also takes place in the cells within an animal’s body, such as a fly or a mouse. DOI: http://dx.doi.org/10.7554/eLife.18124.002

During metastasis, cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I fibers. Cell migration on 2D is initiated by protrusion of the cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM). Cells then move forwards by exerting traction forces on the adhesions at its front and by disassembling adhesions at the rear. In 2D, only the ventral surface of a migrating cell is in contact with the ECM, where cell–matrix adhesions are assembled. In 3D matrices, even though the whole surface of a migrating cell is available for interacting with the ECM, it is unclear whether discrete adhesion structures actually exist. Using high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I, polymerized at a slow rate, to allow the formation of a network that resembles the organization of EMC observed in vivo. Vinculin aggregates were detected in the cellular protrusions, frequently colocalizing with collagen fibers, implying they correspond to adhesion structures in 3D. As the distance from the substrate bottom increases, adhesion aggregates become smaller and almost undetectable in some cell lines. Using intravital imaging we show here, for the first time, the existence of adhesome proteins aggregates in vivo. These aggregates share similarities with the ones found in 3D collagen matrices. It still remains to be determined if adhesions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D. This will provide a major step forward in understanding cell migration in more physiological environments.

Adhesion between cardiac myocytes is essential for the heart to function as an electromechanical syncytium. Although cell-matrix and cell–cell adhesions reorganize during development and disease, the hierarchical cooperation between these subcellular structures is poorly understood. We reasoned that, during cardiac development, focal adhesions mechanically stabilize cells and tissues during myofibrillogenesis and intercalated disc assembly. As the intercalated disc matures, we postulated that focal adhesions disassemble as systolic stresses are transmitted intercellularly. Finally, we hypothesized that pathological remodeling of cardiac microenvironments induces excessive mechanical loading of intercalated discs, leading to assembly of stabilizing focal adhesions adjacent to the junction. To test our model, we engineered μtissues composed of two ventricular myocytes on deformable substrates of tunable elasticity to measure the dynamic organization and functional remodeling of myofibrils, focal adhesions, and intercalated discs as cooperative ensembles. Maturing μtissues increased systolic force while simultaneously developing into an electromechanical syncytium by disassembling focal adhesions at the cell–cell interface and forming mature intercalated discs that transmitted the systolic load. We found that engineering the microenvironment to mimic fibrosis resulted in focal adhesion formation adjacent to the cell–cell interface, suggesting that the intercalated disc required mechanical reinforcement. In these pathological microenvironments, μtissues exhibited further evidence of maladaptive remodeling, including lower work efficiency, longer contraction cycle duration, and weakened relationships between cytoskeletal organization and force generation. These results suggest that the cooperative balance between cell-matrix and cell–cell adhesions in the heart is guided by an architectural and functional hierarchy established during development and disrupted during disease.

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

Cell migration is a crucial event for tissue organization during development, and its dysregulation leads to several diseases, including cancer. Cells exhibit various types of migration, such as single mesenchymal or amoeboid migration, collective migration and scaffold cell-dependent migration. The migration properties are partly dictated by cell adhesion and its endocytic regulation. While an epithelial-mesenchymal transition (EMT)-mediated mesenchymal cell migration requires the endocytic recycling of integrin-mediated adhesions after the disruption of cell-cell adhesions, an amoeboid migration is not dependent on any adhesions to extracellular matrix (ECM) or neighboring cells. In contrast, a collective migration is mediated by both cell-cell and cell-ECM adhesions, and a scaffold cell-dependent migration is regulated by the endocytosis and recycling of cell-cell adhesion molecules. Although some invasive carcinoma cells exhibit an EMT-mediated mesenchymal or amoeboid migration, other cancer cells are known to maintain cadherin-based cell-cell adhesions and epithelial morphology during metastasis. On the other hand, a scaffold cell-dependent migration is mainly utilized by migrating neurons in normal developing brains. This review will summarize the structures of cell adhesions, including adherens junctions and focal adhesions, and discuss the regulatory mechanisms for the dynamic behavior of cell adhesions by endocytic pathways in cell migration in physiological and pathological conditions, focusing particularly on neural development and cancer metastasis.

Summary Background Epithelial tissues undergo extensive collective movements during morphogenesis, repair, and renewal. Collective epithelial cell migration requires the intercellular coordination of cell-cell adhesions and the establishment of anterior-posterior polarity, while maintaining apical-basal polarity, but how this is achieved at the molecular level is not well understood. Results Using an RNA interference-based screen to identify Rho family GTPase regulators required for the collective migration of human bronchial epithelial cells, we identified myosin-IXA (gene name: Myo9a ). Depletion of myosin-IXA, a RhoGAP and actin motor protein, in collectively migrating cells led to altered organization of the actin cytoskeleton and tension-dependent disruption of cell-cell adhesions, followed by an inability to form new adhesions resulting in cell scattering. Closer examination revealed that myosin-IXA is required during the formation of junction-associated actin bundles soon after cell-cell contact. Structure-function analysis of myosin-IXA revealed that the motor domain is necessary and sufficient for binding to actin filaments, whereas expression of the RhoGAP domain partially rescued the cell scattering phenotype induced by myosin-IXA depletion. Finally, a fluorescence resonance energy transfer biosensor revealed a significant increase in Rho activity at nascent cell-cell contacts in myosin-IXA depleted cells compared to controls. Conclusion We propose that myosin-IXA locally regulates Rho and the assembly of thin actin bundles associated with nascent cell-cell adhesions and that this is required to sustain the collective migration of epithelial cells.

Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force.

Lamellar actin architecture at adhesion sites may serve as a structural template that facilitates focal adhesion maturation over a wide range of tension., Focal adhesion composition and size are modulated in a myosin II–dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions. Despite relatively unperturbed adhesion dynamics and force transmission, impaired stress fiber assembly impeded focal adhesion compositional maturation and fibronectin remodeling. Finally, we show that compositional maturation of focal adhesions could occur even when myosin II–dependent cellular tension was reduced by 80%. We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions. This work identifies the essential role of lamellar actin architecture in adhesion maturation.

The adaptor protein APPL1 regulates cell migration and adhesion dynamics by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration., Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.

Cadherin-based intercellular adhesions are important determinants of proper tissue architecture. These adhesions must be both stable and dynamic to maintain tissue integrity as cells undergo morphogenetic movements during development. The role of α-catenin in this process has been vigorously debated due to conflicting in vitro and in vivo evidence regarding its molecular mechanism of action. Recent data supports the classical view that α-catenin facilitates actin attachments at adherens junctions, but also suggests that α-catenin may act as a force transducer, and may have additional roles in the cytoplasm. These multiple functions for α-catenin converge on the regulation of adhesion and may help to explain its stable yet dynamic nature.

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.

Highlights ► The surface of endosomes provides a stage to assemble signaling complexes, to transport signaling molecules and to modify and terminate signal transduction. ► Several important signaling molecules, including RhoGTPases, Src, and MAPK were shown to utilize signaling endosomes to regulate cell migration., Cell migration is a complex biological process that is under the tight control of diverse signaling events. While many of the involved signaling molecules diffuse rapidly within cells, it now seems that certain key regulators of cell migration prefer to travel on endosomes. In this review we will discuss the multiple roles of signaling endosomes in regulation of local migration stimuli, dynamics of focal adhesions, cell contractility and locomotion.

The physical linkage between the extracellular matrix and the actin cytoskeleton of a cell is made by structures known as focal adhesions, acting through integrin receptors. They are of fundamental importance in human physiology because they mediate cell adhesion, mechanosensing and signalling for the control of cell growth and differentiation. The molecular architecture of focal adhesions has now been determined using three-dimensional super-resolution fluorescence microscopy to map protein organization at the nanoscale level. They are revealed as well-organized ultrastructures in which integrins and actin are separated by a 40-nanometre-long core consisting of partially overlapping protein-specific layers, spanned by talin tethers. The multilaminar architecture creates three or more separate compartments that mediate the interdependent functions of focal adhesions. Focal adhesions link the extracellular matrix by integrin receptors to cytoplasmic actin filaments and are fundamental to human physiology. These authors determine the molecular architecture of focal adhesions by mapping protein organization at the nanoscale level. The results demonstrate that focal adhesions possess a well-organized ultrastructure made up of at least three spatial and functional compartments that mediate their interdependent functions. Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing1,2. Focal adhesions are multifunctional organelles that mediate cell–ECM adhesion, force transmission, cytoskeletal regulation and signalling1,2,3. Focal adhesions consist of a complex network4 of trans-plasma-membrane integrins and cytoplasmic proteins that form a

Adhesions are a central mechanism by which cells mechanically interact with the surrounding extracellular matrix (ECM) and neighboring cells. In both cell-ECM and cell-cell adhesions, forces generated within the actin cytoskeleton are transmitted to the surrounding environment and are essential for numerous morphogenic processes. Despite differences in many molecular components that regulate cell-cell and cell-ECM adhesions, the roles of F-actin dynamics and mechanical forces in adhesion regulation are surprisingly similar. Moreover, force transmission at adhesions occurs concomitantly with dynamic F-actin; proteins comprising the adhesion of F-actin to the plasma membrane must accommodate this movement while still facilitating force transmission. Thus, despite different molecular architectures, integrin and cadherin-mediated adhesions operate with common biophysical characteristics to transmit and respond to mechanical forces in a multicellular tissue.

Summary Background The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. Although adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown. Results To elucidate the nature of force transmission as myosin II tension is applied to focal adhesions, we correlated the dynamics of focal adhesion proteins and the actin cytoskeleton to local traction stresses. Under low extracellular tension, newly formed adhesions near the cell periphery underwent a transient retrograde displacement preceding elongation. We found that myosin II-generated tension drives this mobility, and we determine the interface of differential motion, or "slip," to be between integrin and the ECM. The magnitude and duration of both adhesion slip and associated F-actin dynamics is strongly modulated by ECM compliance. Traction forces are generated throughout the slip period, and adhesion immobilization occurs at a constant tension. Conclusions We have identified a tension-dependent, extracellular "clutch" between integrins and the extracellular matrix; this clutch stabilizes adhesions under myosin II driven tension. The current work elucidates a mechanism by which force transmission is modulated during focal adhesion maturation.

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is approximately 2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

Dystroglycan is a ubiquitously expressed cell adhesion protein. Its principal role has been determined as a component of the dystrophin-glycoprotein complex of muscle, where it constitutes a key component of the costameric cell adhesion system. To investigate more fundamental aspects of dystroglycan function in cell adhesion, we examined the role of dystroglycan in the dynamics and assembly of cellular adhesions in myoblasts. We show that β-dystroglycan is recruited to adhesion structures and, based on staining for vinculin, that overexpression or depletion of dystroglycan affects both size and number of fibrillar adhesions. Knockdown of dystroglycan increases the size and number of adhesions, whereas overexpression decreases the number of adhesions. Dystroglycan knockdown or overexpression affects the ability of cells to adhere to different substrates, and has effects on cell migration that are consistent with effects on the formation of fibrillar adhesions. Using an SH3 domain proteomic screen, we identified vinexin as a binding partner for dystroglycan. Furthermore, we show that dystroglycan can interact indirectly with vinculin by binding to the vinculin-binding protein vinexin, and that this interaction has a role in dystroglycan-mediated cell adhesion and spreading. For the first time, we also demonstrate unequivocally that β-dystroglycan is a resident of focal adhesions.

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.

There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1-3 or deleted in liver cancer 1-3 (DLC1-3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265-459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.

International audience; Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.

Using two-colour imaging and high resolution TIRF microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the nascent adhesions either disassemble or mature through growth and elongation. Maturation occurs along an alpha-actinin-actin template that elongates centripetally from nascent adhesions. Alpha-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin crosslinking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin-bound but motor-inhibited mutant of myosin IIA shows that the actin crosslinking function of myosin II mediates initial adhesion maturation. From these studies, we have developed a model for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization.