RESUMEN
Cell adhesions link cells to the extracellular matrix (ECM) and to each other and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping, functional modules. These modules establish physical associations with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as to sense and translate the mechanical properties of the cellular environment into changes in cell organization and behavior. Here, we review the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions and how adhesion molecules mediate cross talk between cell-ECM and cell-cell adhesion sites.
Asunto(s)
Actinas/fisiología , Adhesión Celular , Comunicación Celular , Matriz Extracelular/metabolismo , Actinas/metabolismo , Mecanotransducción Celular , Transducción de SeñalRESUMEN
Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.
Asunto(s)
Carcinoma de Células Escamosas/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Fibronectinas/farmacología , Neoplasias de la Boca/patología , Transducción de Señal/efectos de los fármacos , Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias de la Boca/metabolismoRESUMEN
SUMMARY: Stimuli that promote cell migration, such as chemokines, cytokines, and growth factors in metazoans and cyclic AMP in Dictyostelium, activate signaling pathways that control organization of the actin cytoskeleton and adhesion complexes. The Rho-family GTPases are a key convergence point of these pathways. Their effectors include actin regulators such as formins, members of the WASP/WAVE family and the Arp2/3 complex, and the myosin II motor protein. Pathways that link to the Rho GTPases include Ras GTPases, TorC2, and PI3K. Many of the molecules involved form gradients within cells, which define the front and rear of migrating cells, and are also established in related cellular behaviors such as neuronal growth cone extension and cytokinesis. The signaling molecules that regulate migration can be integrated to provide a model of network function. The network displays biochemical excitability seen as spontaneous waves of activation that propagate along the cell cortex. These events coordinate cell movement and can be biased by external cues to bring about directed migration.
Asunto(s)
Quimiotaxis , Transducción de Señal , Animales , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front-back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front-back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.
Asunto(s)
Sinapsis/enzimología , Quinasas Asociadas a rho/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Actomiosina/metabolismo , Actomiosina/ultraestructura , Animales , Células CHO , Adhesión Celular , Movimiento Celular , Polaridad Celular , Cricetinae , Cricetulus , Espinas Dendríticas/enzimología , Espinas Dendríticas/ultraestructura , Humanos , Ratones , Transporte de Proteínas , Ratas , Sinapsis/ultraestructuraRESUMEN
In this study, we show that the role of nonmuscle myosin II (NMII)-B in front-back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935-1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B-like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.
Asunto(s)
Polaridad Celular , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo IIB no Muscular/fisiología , Actomiosina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Adhesión Celular , Movimiento Celular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Miosina Tipo IIB no Muscular/química , Estabilidad Proteica , Estructura Terciaria de ProteínaRESUMEN
Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.
Asunto(s)
Actinina/fisiología , Espinas Dendríticas/metabolismo , Hipocampo/citología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Espinas Dendríticas/ultraestructura , Transporte de Proteínas , RatasRESUMEN
We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time-frequency (ν, nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for STICS that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins (α6- and αLß2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and ICAM ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrinligand interaction, along with the internal myosin-motor generated force, varies for different integrin-ligand pairs, consistent with previous results.
RESUMEN
CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation, and decreased cell migration, correlating with increased active Rac. Reexpression of wild-type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell-permeable peptides.
Asunto(s)
Movimiento Celular , Tetraspanina 28/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Aminoácidos , Antígenos CD59/metabolismo , Adhesión Celular , Activación Enzimática , Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Tetraspanina 24/metabolismo , Tetraspanina 28/química , Tetraspanina 28/genética , Imagen de Lapso de TiempoRESUMEN
Recognition of the importance of cell adhesion grew steadily during the twentieth century as it promised answers to fundamental questions in diverse fields that included cell biology, developmental biology, tumorigenesis, immunology and neurobiology. However, the route towards a better understanding of its molecular basis was long and difficult, with many false starts. Major progress was made in the late 1970s to late 1980s with the identification of the major families of adhesion molecules, including integrins and cadherins. This in turn set the stage for the explosive growth in adhesion research over the past 25 years.
Asunto(s)
Moléculas de Adhesión Celular/historia , Animales , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/fisiología , Proteínas de la Matriz Extracelular/historia , Proteínas de la Matriz Extracelular/fisiología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteínas de la Membrana/historia , Proteínas de la Membrana/fisiología , RatonesRESUMEN
Two-color spatio-temporal image cross-correlation spectroscopy (STICCS) is a new, to our knowledge, image analysis method that calculates space-time autocorrelation and cross-correlation functions from fluorescence intensity fluctuations. STICCS generates cellular flow and diffusion maps that reveal interactions and cotransport of two distinct molecular species labeled with different fluorophores. Here we use computer simulations to map the capabilities and limitations of STICCS for measurements in complex heterogeneous environments containing micro- and macrostructures. We then use STICCS to analyze the co-flux of adhesion components in migrating cells imaged using total internal reflection fluorescence microscopy. The data reveal a robust, time-dependent co-fluxing of certain integrins and paxillin in adhesions in protrusions when they pause, and in adhesions that are sliding and disassembling, demonstrating that the molecules in these adhesions move as a complex. In these regions, both α6ß1- or αLß2-integrins, expressed in CHO.B2 cells, co-flux with paxillin; an analogous cotransport was seen for α6ß1-integrin and α-actinin in U2OS. This contrasts with the behavior of the α5ß1-integrin and paxillin, which do not co-flux. Our results clearly show that integrins can move in complexes with adhesion proteins in protrusions that are retracting.
Asunto(s)
Movimiento Celular , Matriz Extracelular/metabolismo , Espectrometría de Fluorescencia/métodos , Actinina/metabolismo , Animales , Células CHO , Adhesión Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Humanos , Integrinas/metabolismo , Microscopía Fluorescente , Modelos Teóricos , Paxillin/metabolismoRESUMEN
Adhesive and migratory behavior can be cell type, integrin, and substrate dependent. We have compared integrin and substrate differences using three integrin receptors: α5ß1, α6ß1, and αLß2 expressed in a common cell type, CHO.B2 cells, which lack integrin α subunits, as well as in different cell types that express one or more of these integrins. We find that CHO.B2 cells expressing either α6ß1 or αLß2 integrins migrate and protrude faster and are more directionally persistent on laminin or ICAM-1, respectively, than CHO.B2 cells expressing α5ß1 on fibronectin. Despite rapid adhesion maturation and the presence of large adhesions in both the α6ß1- and αLß2-expressing cells, they display robust tyrosine phosphorylation. In addition, whereas myosin II regulates adhesion maturation and turnover, protrusion rates, and polarity in cells migrating on fibronectin, surprisingly, it does not have comparable effects in cells expressing α6ß1 or αLß2. This apparent difference in the integration of myosin II activity, adhesion, and migration arises from alterations in the ligand-integrin-actin linkage (molecular clutch). The elongated adhesions in the protrusions of the α6ß1-expressing cells on laminin or the αLß2-expressing cells on ICAM-1 display a novel, rapid retrograde flux of integrin; this was largely absent in the large adhesions in protrusions of α5ß1-expressing cells on fibronectin. Furthermore, the force these adhesions exert on the substrate in protrusive regions is reduced compared to similar regions in α5-expressing cells, and the adhesion strength is reduced. This suggests that intracellular forces are not efficiently transferred from actomyosin to the substratum due to altered adhesion strength, that is, avidity, affinity, or the ligand-integrin-actin interaction. Finally, we show that the migration of fast migrating leukocytes on fibronectin or ICAM-1 is also largely independent of myosin II; however, their adhesions are small and do not show retrograde fluxing suggesting other intrinsic factors determine their migration differences.
Asunto(s)
Adhesión Celular , Movimiento Celular , Integrinas/metabolismo , Ligandos , Animales , Células CHO , Adhesión Celular/genética , Línea Celular , Movimiento Celular/genética , Extensiones de la Superficie Celular/genética , Extensiones de la Superficie Celular/metabolismo , Cricetinae , Fibronectinas/metabolismo , Expresión Génica , Células HL-60 , Humanos , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/metabolismo , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Integrinas/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Laminina/metabolismo , Leucocitos/metabolismo , Miosina Tipo II/metabolismo , Unión Proteica , Multimerización de Proteína , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Uniones Célula-Matriz/química , Uniones Célula-Matriz/metabolismo , Actinas/metabolismo , Animales , Comunicación Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Humanos , Mecanotransducción Celular , Microscopía Electrónica/métodos , Complejos Multiproteicos , Transducción de Señal , Tomografía/métodosAsunto(s)
Adhesión Celular , Adhesiones Focales/química , Actinas/química , Actinas/fisiología , Animales , Diferenciación Celular , Movimiento Celular , Microambiente Celular , Matriz Extracelular/química , Matriz Extracelular/fisiología , Colágenos Fibrilares/química , Adhesiones Focales/fisiología , Humanos , Integrinas/química , Integrinas/fisiología , PolimerizacionRESUMEN
Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.
Asunto(s)
Espinas Dendríticas/ultraestructura , Miosina Tipo IIB no Muscular/metabolismo , Densidad Postsináptica/ultraestructura , Animales , Extensiones de la Superficie Celular/ultraestructura , Espinas Dendríticas/metabolismo , Fosforilación , Densidad Postsináptica/metabolismo , Seudópodos , Ratas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Integrin-based adhesion has served as a model for studying the central role of adhesion in migration. In this article, we outline modes of migration, both integrin-dependent and -independent in vitro and in vivo. We next discuss the roles of adhesion contacts as signaling centers and linkages between the ECM and actin that allows adhesions to serve as traction sites. This includes signaling complexes that regulate migration and the interplay among adhesion, signaling, and pliability of the substratum. Finally, we address mechanisms of adhesion assembly and disassembly and the role of adhesion in cellular polarity.
Asunto(s)
Movimiento Celular/fisiología , Polaridad Celular/fisiología , Adhesiones Focales/fisiología , Integrinas/metabolismo , Modelos Biológicos , Transducción de Señal/fisiologíaRESUMEN
A new study shows that protein kinase A (PKA) activity establishes a signaling loop that governs protrusion-retraction cycles in migrating cells. PKA activity near the leading edge of protrusions phosphorylates RhoA and inhibits its activity via increased association with RhoGDI.
Asunto(s)
Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , FosforilaciónRESUMEN
Cell migration is a fundamental process that controls morphogenesis and inflammation. Its deregulation causes or is part of many diseases, including autoimmune syndromes, chronic inflammation, mental retardation, and cancer. Cell migration is an integral part of the cell biology, embryology, immunology, and neuroscience fields; as such, it has benefited from quantum leaps in molecular biology, biochemistry, and imaging techniques, and the emergence of the genomic and proteomic era. Combinations of these techniques have revealed new and exciting insights that explain how cells adhere and move, how the migration of multiple cells are coordinated and regulated, and how the cells interact with neighboring cells and/or react to changes in their microenvironment. This introduction provides a primer of the molecular and cellular insights, particularly the signaling networks, which control the migration of individual cells as well as collective migrations. The rest of the chapters are devoted to describe in detail some of the most salient technical advances that have illuminated the field of cell migration in recent years.
Asunto(s)
Actinas/metabolismo , Movimiento Celular , Miosinas/metabolismo , Animales , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Extensiones de la Superficie Celular/metabolismo , Uniones Célula-Matriz/metabolismo , Humanos , Transducción de SeñalRESUMEN
Migratory front-back polarity emerges from the cooperative effect of myosin IIA (MIIA) and IIB (MIIB) on adhesive signaling. We demonstrate here that, during polarization, MIIA and MIIB coordinately promote localized actomyosin bundling, which generates large, stable adhesions that do not signal to Rac and thereby form the cell rear. MIIA formed dynamic actomyosin proto-bundles that mark the cell rear during spreading; it also bound to actin filament bundles associated with initial adhesion maturation in protrusions. Subsequent incorporation of MIIB stabilized the adhesions and actomyosin filaments with which it associated and formed a stable, extended rear. These adhesions did not turn over and no longer signal to Rac. Microtubules fine-tuned the polarity by positioning the front opposite the MIIA/MIIB-specified rear. Decreased Rac signaling in the vicinity of the MIIA/MIIB-stabilized proto-bundles and adhesions was accompanied by the loss of Rac guanine nucleotide exchange factor (GEFs), like ßPIX and DOCK180, and by inhibited phosphorylation of key residues on adhesion proteins that recruit and activate Rac GEFs. These observations lead to a model for front-back polarity through local GEF depletion.
Asunto(s)
Movimiento Celular , Polaridad Celular , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Transducción de Señal , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animales , Células CHO , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Cricetinae , Cricetulus , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosforilación , Unión ProteicaRESUMEN
We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.