Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Pasapera, Ana M.; Schneider, Ian C.; Rericha, Erin; Schlaepfer, David D.; Waterman‐Storer, Clare M.

doi:10.1083/jcb.200906012

Cited by 502 publications

(587 citation statements)

References 62 publications

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“…Transmission of force from the actin cortex to the cilium might occur through simple steric interactions or specific connections to the cytoskeleton through striated rootlets (20) or basal bodyassociated focal adhesion proteins (39,45). The hinging degree of freedom could be important for other cellular processes, such as ciliary orientation in migrating fibroblasts, which have been found to align their cilia in their direction of motion (4).…”

Section: Discussionmentioning

confidence: 99%

Intracellular and extracellular forces drive primary cilia movement

Battle

Ott

Burnette

et al. 2015

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

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Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.

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Section: Discussionmentioning

confidence: 99%

Intracellular and extracellular forces drive primary cilia movement

Battle

Ott

Burnette

et al. 2015

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

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“…Pxn is used as the FA marker in this study, as its abundance in FAs is largely independent from myosin II activity 65 . This was used to generate a spatial mask to quantify and normalize local FAK activation as a function of myosin II activity, as has been done in previous studies 66,67 . An in-house built MATLAB script was used to quantify micrographs based on the morphometry and intensity of adhesions.…”

Section: Methodsmentioning

confidence: 99%

Dynamic catch of a Thy-1–α5β1+syndecan-4 trimolecular complex

Fiore

Chen

et al. 2014

Nat Commun

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Cancer cell adhesion to the vascular endothelium is a critical step of tumour metastasis. Endothelial surface molecule Thy-1 (CD90) is implicated in the metastatic process through its interactions with integrins and syndecans. However, how Thy-1 supports cell-cell adhesion in a dynamic mechanical environment is not known. Here we show that Thy-1 supports b 1 integrin-and syndecan-4 (Syn4)-mediated contractility-dependent mechanosignalling of melanoma cells. At the single-molecule level, Thy-1 is capable of independently binding a 5 b 1 integrin and syndecan-4 (Syn4) receptors. However, in the presence of both a 5 b 1 and Syn4, the two receptors bind cooperatively to Thy-1, to form a trimolecular complex. This trimolecular complex displays a unique phenomenon we coin 'dynamic catch', characterized by abrupt bond stiffening followed by the formation of catch bonds, where force prolongs the bond lifetime. Thus, we reveal a new class of trimolecular interactions where force strengthens the synergistic binding of two co-receptors and modulates downstream mechanosignalling.

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“…Furthermore, phosphorylation of FAK at Tyr 397 is required for invadopodia formation for breast cancer cells (25), and FAK PY397 is differentially expressed in metastatic cervical carcinoma relative to noncancerous epithelium (26). FAK is required for mechanical stress-induced cell polarization (6,27), and the FAK-paxillin-vinculin signaling complex is required for rigidity sensing and durotaxis (9). To quantify the localization of FA proteins, we defined a normalized metric to measure the distribution of fluorescently tagged proteins at the cell membrane.…”

Section: Significancementioning

confidence: 99%

“…Paxillin is required for stiffness mechanosensing (6), protrusion formation at regions of high stress (8), and durotaxis (9). To evaluate the role of paxillin in upstream FA polarization and rheotaxis, we treated cells with paxillin siRNA, resulting in knockdown of paxillin expression by 80% as confirmed by immunoblotting (Fig.…”

Section: Paxillin Is Required For Upstream Actin Polarization and Promentioning

confidence: 99%

“…The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK-paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin-a key mechanosensor (7)-mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK-paxillin-vinculin signaling is required for mechanosensing and durotaxis (9).…”

mentioning

confidence: 99%

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Mechanotransduction of fluid stresses governs 3D cell migration

Polacheck

German

Mammoto

et al. 2014

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

231

215

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Solid tumors are characterized by high interstitial fluid pressure, which drives fluid efflux from the tumor core. Tumor-associated interstitial flow (IF) at a rate of ∼3 μm/s has been shown to induce cell migration in the upstream direction (rheotaxis). However, the molecular biophysical mechanism that underlies upstream cell polarization and rheotaxis remains unclear. We developed a microfluidic platform to investigate the effects of IF fluid stresses imparted on cells embedded within a collagen type I hydrogel, and we demonstrate that IF stresses result in a transcellular gradient in β1-integrin activation with vinculin, focal adhesion kinase (FAK), FAK PY397 , F actin, and paxillindependent protrusion formation localizing to the upstream side of the cell, where matrix adhesions are under maximum tension. This previously unknown mechanism is the result of a force balance between fluid drag on the cell and matrix adhesion tension and is therefore a fundamental, but previously unknown, stimulus for directing cell movement within porous extracellular matrix.mechanobiology | breast cancer | metastasis I ntegrins and associated focal adhesion (FA) proteins form a tension-sensitive mechanical link between the extracellular matrix (ECM) and the cytoskeleton, and serve as key components in the signaling cascade by which cells transduce mechanical signals into biological responses (mechanotransduction) (1, 2). Contractile stresses generated by the cell are balanced by tractions at cell-substrate adhesions, and the FA protein vinculin accumulates at regions of high substrate stress (3, 4). The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK-paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin-a key mechanosensor (7)-mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK-paxillin-vinculin signaling is required for mechanosensing and durotaxis (9).The tumor microenvironment imparts mechanical and chemical signals on tumor and stromal cells (10), and advanced breast carcinomas are characterized by high interstitial fluid pressure (11), an indicator of poor prognosis (12). This elevated fluid pressure drives interstitial flow (IF) and alters chemical transport within the tumor (13), and IF influences tumor cell migration through the generation of autocrine chemokine gradients (14). Equally important, although not as well understood, is the physical drag imparted on the ECM and constitutive cells (15) by IF, which is analogous to the FA-activating shear stresses generated on endothelial cells by hemodynamic forces (16). With endothelial cells, shear stress can be the dominant mechanical stimulus that induces FAK activation and cytoskeletal remodeling; however, for cells embedded within a porous matrix scaffold, the ratio of the force due to the pressure drop across the cell to the total shear force is inversely proportional to hydrogel p...

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Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Abstract: FAK-mediated myosin-dependent paxillin phosphorylation is necessary to bring vinculin to maturing focal adhesions, reinforcing the link between the cytoskeleton and the ECM.

Cited by 502 publications

References 62 publications

Intracellular and extracellular forces drive primary cilia movement

Intracellular and extracellular forces drive primary cilia movement

Dynamic catch of a Thy-1–α5β1+syndecan-4 trimolecular complex

Mechanotransduction of fluid stresses governs 3D cell migration

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