RESUMO
Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Movimento Celular , Proteínas do Citoesqueleto , Ligação Proteica , Talina , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Adesões Focais/metabolismo , Integrinas/metabolismo , Células MCF-7 , Microtúbulos/metabolismo , Fosforilação , Talina/metabolismoRESUMO
The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5ß1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
Assuntos
Fibronectinas , Adesões Focais , Talina , Tensinas , Adesão Celular , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Integrinas/metabolismo , Talina/genética , Talina/metabolismo , Tensinas/genética , Tensinas/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Integrins mediate cell adhesion by connecting the extracellular matrix to the intracellular cytoskeleton and orchestrate signal transduction in response to chemical and mechanical stimuli by interacting with many cytoplasmic proteins. We used BioID to interrogate the interactomes of ß1 and ß3 integrins in epithelial cells and identified PEAK1 as an interactor of the RGD-binding integrins α5ß1, αVß3, and αVß5 in focal adhesions. We demonstrate that the interaction between integrins and PEAK1 occurs indirectly through Tensin3, requiring both the membrane-proximal NPxY motif on the integrin ß tail and binding of the SH2 domain of Tensin3 to phosphorylated Tyr-635 on PEAK1. Phosphorylation of Tyr-635 is mediated by Src and regulates cell migration. Additionally, we found that Shc1 localizes in focal adhesions in a PEAK1 phosphorylated Tyr-1188-dependent fashion. Besides binding Shc1, PEAK1 also associates with a protein cluster that mediates late EGFR/Shc1 signaling. We propose a model in which PEAK1 binds Tensin3 and Shc1 to converge integrin and growth factor receptor signal transduction.
Assuntos
Adesão Celular , Integrinas , Proteínas Tirosina Quinases , Tensinas , Movimento Celular , Adesões Focais/metabolismo , Humanos , Integrina beta3/metabolismo , Integrinas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Tensinas/metabolismoRESUMO
Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10-12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.
Assuntos
Axônios/fisiologia , Matriz Extracelular/metabolismo , Mecanotransdução Celular , Crescimento Neuronal , Neurônios/citologia , Vinculina/metabolismo , Animais , Adesão Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais , Integrinas/genética , Integrinas/metabolismo , Camundongos , Neurônios/metabolismo , Vinculina/genéticaRESUMO
Talin, vinculin, and paxillin are core components of the dynamic link between integrins and actomyosin. Here, we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are autoinhibited and do not interact with each other. However, contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of autoinhibition is sufficient to mediate their tight interaction. We also found that paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrated that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force machinery and focal adhesion maturation.
Assuntos
Fibroblastos/metabolismo , Adesões Focais/fisiologia , Paxilina/fisiologia , Estresse Mecânico , Talina/antagonistas & inibidores , Vinculina/fisiologia , Citoesqueleto de Actina , Animais , Células Cultivadas , Fibroblastos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Talina/metabolismoRESUMO
Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.
Assuntos
Proteínas da Matriz Extracelular/química , Matriz Extracelular/química , Lasers , Sistemas Microeletromecânicos/métodos , Impressão Tridimensional , Adsorção , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Sistemas Microeletromecânicos/instrumentação , Impressão Tridimensional/instrumentação , Reprodutibilidade dos TestesRESUMO
Cell division involves the tightly coordinated rearrangement of actin and microtubules (MTs). We have previously shown that a member of the family of growth arrest-specific 2-like proteins, GAS2-like 1 (G2L1) regulates actin-MT crosstalk through its associations with plus-end microtubule tip-binding (EB) proteins. Here we show that G2L1 is involved in the regulation of cell division. We show that the depletion of G2L1 results in a reduction in the number of cells undergoing cell division and a significant proportion of those cells that do divide are either multinucleated, display deformed nuclei, or undergo cell division at a much slower rate. Exogenous expression of G2L1 mutants revealed that the association of G2L1 with EB1 is critical for regulated cell division and blocking this interaction inhibits cell division as observed in cells lacking G2L1. Taken together, our data suggest that G2L1 controls the precise regulation and successful progression of cell division through its binding to EB-proteins.
Assuntos
Divisão Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Divisão Celular/genética , Linhagem Celular Tumoral , Humanos , Proteínas dos Microfilamentos/genética , Microtúbulos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.
Assuntos
Movimento Celular/efeitos da radiação , Neuropeptídeos/metabolismo , Vinculina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Endocitose/fisiologia , Endocitose/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Ondas Ultrassônicas , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Genetic mutations in the SHANK family of proteins are linked to multiple neuropsychiatric disorders including autism spectrum disorders. A study now elucidates critical roles for SHANK in regulating integrin-mediated cell-extracellular matrix adhesion, by sequestering integrin activators.
Assuntos
Adesão Celular , Talina/genética , Matriz Extracelular , Humanos , Integrinas/genéticaRESUMO
Focal adhesions (FAs) are macromolecular complexes that regulate cell adhesion and mechanotransduction. By performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoactivation (FLAP) experiments, we found that the mobility of core FA proteins correlates with their function. Structural proteins such as tensin, talin and vinculin are significantly less mobile in FAs than signaling proteins such as FAK (also known as PTK2) and paxillin. The mobilities of the structural proteins are directly influenced by substrate stiffness, suggesting that they are involved in sensing the rigidity of the extracellular environment. The turnover rates of FAK and paxillin, as well as kindlin2 (also known as FERMT2), are not influenced by substrate stiffness. By using specific Src and FAK inhibitors, we reveal that force-sensing by vinculin occurs independently of FAK and paxillin phosphorylation. However, their phosphorylation is required for downstream Rac1-driven cellular processes, such as protrusion and cell migration. Overall, we show that the FA is composed of different functional modules that separately control mechanosensing and the cellular mechano-response.
Assuntos
Adesões Focais/metabolismo , Mecanotransdução Celular , Animais , Movimento Celular , Matriz Extracelular/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Paxilina/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Transporte Proteico , Pseudópodes/metabolismo , Transdução de Sinais , Vinculina/metabolismo , Quinases da Família src/metabolismoRESUMO
External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a variety of diseases. Cells contain several types of adhesions, linking them to their external environment. It is through these adhesions that forces are both sensed (from the outside inwards) and applied (from inside to out). Furthermore, several adhesion-based proteins are sensitive to changes in intracellular forces, utilising them for activation and regulation. Here, we outline how vinculin, a key component of integrin-mediated adhesions linking the actin cytoskeleton to the extracellular matrix (ECM), is regulated by force and acts as force transducing protein. We discuss the role of vinculin in vivo and its place in health and disease; summarise the proposed mechanisms by which vinculin is recruited to and activated at integrin-ECM adhesions; and discuss recent findings that place vinculin as the major force sensing and transmitting component of cell-matrix adhesion complexes. Finally, we discuss the role of vinculin in regulating the cellular responses to both the physical properties of the external environment and to externally applied physical stimuli.
Assuntos
Adesão Celular/fisiologia , Integrinas/fisiologia , Vinculina/metabolismo , Matriz Extracelular/fisiologia , Humanos , Modelos Biológicos , Estresse MecânicoRESUMO
The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.