The phospho-caveolin-1 scaffolding domain dampens force fluctuations in focal adhesions and promotes cancer cell migration.
ABSTRACT: Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82-101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD-competing peptides, that Src kinase-dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension, and cancer cell migration is CSD dependent. Quantitative proteomic analysis of Cav1-GST (amino acids 1-101) pull downs showed sixfold-increased binding of vinculin and, to a lesser extent, ?-actinin, talin, and filamin, to phosphomimetic Cav1Y14D relative to nonphosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD-dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high-tension mode, paralleling effects of actin stabilization. This identifies pY14Cav1 as a molecular regulator of focal adhesion tension and suggests that functional interaction between Cav1 Y14 phosphorylation and the CSD promotes focal adhesion traction and, thereby, cancer cell motility.
Project description:Cell-matrix adhesions are central mediators of mechanotransduction, yet the interplay between force and adhesion regulation remains unclear. Here we use live cell imaging to map time-dependent cross-correlations between vinculin-mediated tension and adhesion complex area, revealing a plastic, context-dependent relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions display negative cross-correlation. Furthermore, although large changes in adhesion complex area follow vinculin-mediated tension alterations, small increases in area precede vinculin-mediated tension dynamics. Modelling based on this mapping of the vinculin-mediated tension-adhesion complex area relationship confirms its biological validity, and indicates that this relationship explains adhesion size and lifetime limits, keeping adhesions focal and transient. We also identify a subpopulation of steady-state adhesions whose size and vinculin-mediated tension become stabilized, and whose disassembly may be selectively microtubule-mediated. In conclusion, we define a plastic relationship between vinculin-mediated tension and adhesion complex area that controls fundamental cell-matrix adhesion properties.
Project description:Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.
Project description:Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.
Project description:Caveolin-1 is a transmembrane protein with both tumor promoter and suppressor functions that remain poorly understood. Cav1 phosphorylation by Src kinase on tyrosine 14 is closely associated with focal adhesion dynamics and tumor cell migration, however the role of pCav1 in vivo in tumor progression remains poorly characterized. Herein, we expressed phosphomimetic Y14D, wild type, and non-phosphorylatable Y14F forms of Cav1 in MDA-MB-435 cancer cells. Expression of Cav1Y14D reduced cell proliferation and induced the TP53 tumor suppressor. Ectopic expression in MDA-MB-435 cells of Y14 phosphorylatable Cav1 was required for induction of TP53 in response to oxidative stress. Cav1Y14D promotes an apparent reversal of the Warburg effect and markedly inhibited tumor growth in vivo. However, Cav1 induced pseudopodial recruitment of glycolytic enzymes, and time-lapse intravital imaging showed increased invadopodia protrusion and extravasation into blood vessels for Cav1WT and Y14D but not for Y14F. Our results suggest that Cav1 Y14 phosphorylation levels play a role in the conflicting demands on metabolic resources associated with cancer cell proliferation versus motility.
Project description:Cellular force transmission and mechanotransduction are critical in embryogenesis, normal physiology, and many diseases. Talin plays a key role in these processes by linking integrins to force-generating actomyosin. Using the previously characterized FRET-based talin tension sensor, we observed variations of tension both between and within individual focal adhesions in the same cell. Assembling and sliding adhesions showed gradients with higher talin tension toward the cell center, whereas mature, stable adhesions had uniform talin tension. Total talin accumulation was maximal in high-tension regions; by contrast, vinculin intensity was flat or maximal at the adhesion center, and actin intensity was maximal toward the cell center. To investigate mechanism, we combined talin tension imaging with cellular cryotomography to visualize the correlated actin organization at nanometer resolution. Regions of high talin tension had highly aligned linear actin filaments, whereas regions of low tension had less-well-aligned F-actin. These results reveal an orchestrated spatiotemporal relationship between talin tension, actin/vinculin localization, local actin organization, and focal adhesion dynamics.
Project description:Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The lifecycle of a focal adhesion is a highly coordinated process involving spatial and temporal variations of protein composition, interaction, and cellular tension. The assembly of focal adhesions requires the recruitment and activation of vinculin. Vinculin is present in the cytoplasm in an autoinhibited conformation in which its tail is held pincerlike by its head domains, further stabilized by two high-affinity head-tail interfaces. Vinculin has binding sites for talin and F-actin, but effective binding requires vinculin activation to release its head-tail associations. In migrating cells, it has been shown that the locations of vinculin activation coincide with areas of high cellular tension, and that the highest recorded tensions across vinculin are associated with adhesion assembly. Here, we use a structure-based model to investigate vinculin activation by talin modulated by tensile force generated by transient associations with F-actin. We show that vinculin activation may proceed from an intermediate state stabilized by partial talin-vinculin association. There is a low-force regime and a high-force regime where vinculin activation is dominated by two different pathways with distinct responses to force. Specifically, at zero or low forces, vinculin activation requires substantial destabilization of the main head-tail interface, which is rigid and undergoes very limited fluctuations, despite the other being relatively flexible. This pathway is not significantly affected by force; instead, higher forces favor an alternative pathway, which seeks to release the vinculin tail from its pincerlike head domains before destabilizing the head-tail interfaces. This pathway has a force-sensitive activation barrier and is significantly accelerated by force. Experimental data of vinculin during various stages of the focal adhesion lifecycle are consistent with the proposed force-regulated activation pathway.
Project description:Caveolin-1 (Cav1) is an essential component of caveolae whose Src kinase-dependent phosphorylation on tyrosine 14 (Y14) is associated with regulation of focal adhesion dynamics. However, the relationship between these disparate functions remains to be elucidated. Caveola biogenesis requires expression of both Cav1 and cavin-1, but Cav1Y14 phosphorylation is dispensable. In this paper, we show that Cav1 tyrosine phosphorylation induces caveola biogenesis via actin-dependent mechanotransduction and inactivation of the Egr1 (early growth response-1) transcription factor, relieving inhibition of endogenous<jats:italic>Cav1</jats:italic>and<jats:italic>cavin-1</jats:italic>genes. Cav1 phosphorylation reduces Egr1 binding to Cav1 and cavin-1 promoters and stimulates their activity. In MDA-231 breast carcinoma cells that express elevated levels of Cav1 and caveolae, Egr1 regulated Cav1, and cavin-1 promoter activity was dependent on actin, Cav1, Src, and Rho-associated kinase as well as downstream protein kinase C (PKC) signaling. pCav1 is therefore a mechanotransducer that acts via PKC to relieve Egr1 transcriptional inhibition of Cav1 and cavin-1, defining a novel feedback regulatory loop to regulate caveola biogenesis.
Project description:Caveolin-1 (Cav1) is an essential component of caveolae whose Src kinase-dependent phosphorylation on tyrosine 14 (Y14) is associated with regulation of focal adhesion dynamics. However, the relationship between these disparate functions remains to be elucidated. Caveola biogenesis requires expression of both Cav1 and cavin-1, but Cav1Y14 phosphorylation is dispensable. In this paper, we show that Cav1 tyrosine phosphorylation induces caveola biogenesis via actin-dependent mechanotransduction and inactivation of the Egr1 (early growth response-1) transcription factor, relieving inhibition of endogenous Cav1 and cavin-1 genes. Cav1 phosphorylation reduces Egr1 binding to Cav1 and cavin-1 promoters and stimulates their activity. In MDA-231 breast carcinoma cells that express elevated levels of Cav1 and caveolae, Egr1 regulated Cav1, and cavin-1 promoter activity was dependent on actin, Cav1, Src, and Rho-associated kinase as well as downstream protein kinase C (PKC) signaling. pCav1 is therefore a mechanotransducer that acts via PKC to relieve Egr1 transcriptional inhibition of Cav1 and cavin-1, defining a novel feedback regulatory loop to regulate caveola biogenesis.
Project description: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.
Project description:We present a model to estimate intracellular force variations from live-cell images of actin filament (F-actin) flow during protrusion-retraction cycles of epithelial cells in a wound healing response. To establish a mechanistic relationship between force development and cytoskelal dynamics, force fluctuations were correlated with fluctuations in F-actin turnover, flow and F-actin-vinculin coupling. Our analyses suggest that force transmission at focal adhesions requires binding of vinculin to F-actin and integrin (indirectly), which is modulated at the vinculin-integrin but not the vinculin-F-actin interface. Force transmission at focal adhesions is colocalized in space and synchronized in time with transient increases in the boundary force at the cell edge. Surprisingly, the maxima in adhesion and boundary forces lag behind maximal edge advancement by about 40 s. Maximal F-actin assembly was observed about 20 s after maximal edge advancement. On the basis of these findings, we propose that protrusion events are limited by membrane tension and that the characteristic duration of a protrusion cycle is determined by the efficiency in reinforcing F-actin assembly and adhesion formation as tension increases.