Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

Thievessen, Ingo; Thompson, Peter M.; Berlemont, Sylvain; Plevock, Karen M.; Plotnikov, Sergey V.; Zemljic-Harpf, Alice; Ross, Robert S.; Davidson, Michael W.; Danuser, Gaudenz; Campbell, Sharon L.; Waterman‐Storer, Clare M.

doi:10.1083/jcb.201303129

Cited by 246 publications

(351 citation statements)

References 42 publications

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“…2, we observed a similar hierarchical organization of FA proteins along the z-dimension, with the N-C polarized orientation of talin that seems to span the FA core, linking with actin stress fibers. These results suggest that vinculin is dispensable for the stratified nanoscale architecture of FAs, consistent with previous studies documenting FA assembly and maturation in the absence of vinculin (33,34).…”

Section: Resultssupporting

confidence: 81%

Talin determines the nanoscale architecture of focal adhesions

Liu

Wang

Goh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15°relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.superresolution microscopy | focal adhesions | talin | mechanobiology | nanoscale architecture C ell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2-5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7-10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11-15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins s...

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Section: Resultssupporting

confidence: 81%

Talin determines the nanoscale architecture of focal adhesions

Liu

Wang

Goh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

180

169

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“…Vinculin was shown to be non-uniformly distributed between all three of the axial FA layers, and it was suggested that vinculin "climbs" talin to reach the ARL and bind actin (8). Although numerous observations indicate that vinculin-actin binding is crucial for force transmission and for FA assembly and maturation (50,53), vinculin mutants deficient in actin binding are distributed within the FA structure similarly to wild type (8).…”

Section: Vinculin-lipid-actin Interactions In the Context Of The Fa Tmentioning

confidence: 99%

Mechanisms and Functions of Vinculin Interactions with Phospholipids at Cell Adhesion Sites

Izard

Brown

2016

Journal of Biological Chemistry

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The cytoskeletal protein vinculin is a major regulator of cell adhesion and attaches to the cell surface by binding to specific phospholipids. Structural, biochemical, and biological studies provided much insight into how vinculin binds to membranes, what components it recognizes, and how lipid binding is regulated. Here we discuss the roles and mechanisms of phospholipids in regulating the structure and function of vinculin and of its muscle-specific metavinculin splice variant. A full appreciation of these processes is necessary for understanding how vinculin regulates cell motility, migration, and wound healing, and for understanding of its role in cancer and cardiovascular diseases.Inositol phospholipids are crucial regulators of cell physiology, and their head group interactions play fundamental roles in controlling membrane/cytosol interfaces. In addition to signal transduction at the cell surface, these lipids regulate a range of cellular processes including membrane trafficking, polarity, nuclear events, the permeability and transport functions of membranes, and cytoskeletal organization. Phosphoinositides are spatially and temporally regulated at sites of actin assembly andcytoskeletonremodeling.Phosphatidylinositol4,5-bisphosphate (PIP 2 ) 2 is a precursor of the second messengers diacylglycerol, inositol 3,4,5-trisphosphate, and phosphatidyl 3,4,5-trisphosphate and is crucial in the organization of the actin cytoskeleton at the plasma membrane (1). PIP 2 participates in the recruitment and activation of a wide variety of adaptor proteins and actin regulatory proteins. During the assembly of focal adhesions (FAs), the cytoskeletal protein talin recruits an isoform of phosphatidylinositol 4-phosphate 5-kinase (PIP 5 K␥90) that catalyzes the phosphorylation of phosphatidyl 4-phosphate to generate a local enrichment of PIP 2 (2-4). This localized pool of PIP 2 is critical for the subsequent recruitment of FA proteins and maturation of the FAs (5, 6). Vinculin is an early and essential component of nascent cell-matrix adhesion and acts as a scaffold by binding to several actin-organizing proteins. In mature FAs, vinculin is a key component of the "molecular clutch" that mediates the transmission of force from cytoplasmic F-actin to membrane-bound integrins (7-9). Cytosolic vinculin mainly adopts a closed conformation that is kept inactive through extensive hydrophobic interactions of its globular 91-kDa head (VH, residues 1-840) that harbors binding sites for talin, ␣-actinin, or ␣-catenin to VH (10 -13) and its 21-kDa tail (Vt, residues 879 -1066) domains (14, 15), which are connected via a proline-rich linker (Fig. 1, A and B). The intramolecular VH-Vt interaction masks the binding sites of F-actin (16, 17), PIP 2 (18), and raver1 (19,20) to Vt. Cytoplasmic vinculin must be recruited to FAs and activated to expose ligandbinding sites through a complex process that is not completely understood (21).Vinculin is essential during development due to its role in regulating adhesion and motility, as well as cel...

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“…Indeed, this result is supported by the recent finding by Thievessen, et al that the V T portion of full-length vinculin directly binds to and slows the retrograde flow of actin in mature FAs, thereby establishing distinct lamellipodium-lamellum border and generating high levels of traction force. 28 Somewhat surprisingly, though, this same study also found that FA growth rate correlated with F-actin flow speed independently of vinculin. 28 These findings highlight the need for improving our understanding of the role of the V T in actin binding and bundling.…”

Section: Vinculin Regulates But Is Not Required For the Generation mentioning

confidence: 87%

“…28 Somewhat surprisingly, though, this same study also found that FA growth rate correlated with F-actin flow speed independently of vinculin. 28 These findings highlight the need for improving our understanding of the role of the V T in actin binding and bundling. The V T domain of vinculin first binds to F-actin and then dimerizes to an adjacent F-actin bound V T .…”

Section: Vinculin Regulates But Is Not Required For the Generation mentioning

confidence: 87%

“…Indeed, Thievessen, et al found that FA growth rate and F-actin flow speed-and by inference, cell contractile force-are highly correlated but that vinculin is not required for this coupling. 28 Nevertheless, vinculin does play an important role in modulating FA behavior and force transmission. Indeed, Jano stiak, et al recently showed that Crk-associated substrate (CAS), previously shown to play a key role in transmitting mechanical forces and regulating CSK tension, interacts with the prolinerich strap of vinculin to modulate the dynamics of both proteins and modulate cell stiffness and traction force.…”

Section: Autoinhibitory Head-tail Interaction Regulates the Compositimentioning

confidence: 99%

See 1 more Smart Citation

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

Dumbauld

Garcı́a

2014

Cell Adhesion & Migration

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Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

Abstract: Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

Cited by 246 publications

References 42 publications

Talin determines the nanoscale architecture of focal adhesions

Talin determines the nanoscale architecture of focal adhesions

Mechanisms and Functions of Vinculin Interactions with Phospholipids at Cell Adhesion Sites

A helping hand: How vinculin contributes to cell-matrix and cell-cell force transfer

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