Cells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic vinculin-binding sites, triggers this process. The binding of RIAM to talin could regulate this mechanism. However, the mechanosensitivity of the talin-RIAM complex has never been tested. It is also not known whether RIAM controls the mechanosensitivity of the talin-vinculin complex. To address these issues, we designed an in vitro microscopy assay with purified proteins in which the actomyosin force controls RIAM and vinculin-binding to talin. We demonstrate that actomyosin triggers RIAM dissociation from several talin domains. Actomyosin also provokes the sequential exchange of RIAM for vinculin on talin. The effect of RIAM on this forcedependent binding of vinculin to talin varies from one talin domain to another. This mechanism could allow talin to biochemically code a wide range of forces by selecting different combinations of partners.
Focal adhesions (FAs) mechanically couple the extracellular matrix (ECM) to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis, i.e. modulating integrin activation and controlling actin polymerization, remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin-filament anchoring and thus plays a major role in the establishment of the actin-ECM mechanical coupling. Talin contains three actinbinding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, while the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a Cterminal five-helix-bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in TIRF microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed end elongation while ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, integrinbound talin head retains the ability to inhibit actin assembly.Cell adhesion to the extracellular matrix plays a critical role in many physiological functions such as cell migration, invasion or epithelial basement membrane attachment. Among the multiple adhesion structures, focal adhesions (FAs) play a major role (1,2). These multiprotein complexes couple various extracellular matrices to the actin cytoskeleton via the transmembrane heterodimeric αβ integrins and actin-binding proteins (ABPs) (3,4). The control of actin polymerization by ABPs is thought to play an important role to initiate the formation of the actomyosin stress fibers and control their tension by modulating their elongation. We showed previously that vinculin blocks actin filament barbed end elongation (5), while others reported that VASP promotes the elongation of actin filament barbed ends in a processive-like manner (6,7). Several formins may also play a role in the formation and elongation of Talin head inhibits actin assembly 2 stress fibers (8). However, despite these isolated characterizations, the respective roles of the multiple ABPs and their coordination in this process are poorly understood. The actin-binding protein talin plays a major role in FAs (9) ( Figure S1). First it acts very early to activate integrins. In this process the N-terminal PTB (phosphotyrosine binding) domain, located in the head domain of talin, also known as the F3 subdomain of the ...
The metastatic progression of cancer remains a major issue in patient treatment. Yet, the molecular and cellular mechanisms underlying this process remains unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor prognosis histological form of digestive cancers, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies: MUC CRCs either display a conventional “apical-in” polarity or, more frequently, harbor an inverted “apical-out” topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFb and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, this apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients.
Background Information: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level.Results: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces. Conclusions: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces. Significance: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.
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