Project 1: Vinay Swaminathan The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin. Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NA) within the protrusion to drive leading edge advance. Although the focal adhesion-associated non-receptor tyrosine kinase FAK is known to be required for cell migration through effects on mature focal adhesions, its role in NA formation and lamellipodial dynamics is not clear. Live-cell microscopy of FAK -/- cells with expression of phosphorylation or a FERM domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. We show that phosphorylation on Y397 of FAK promotes dense NA formation, but is dispensible for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK-Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of ECM concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration. A paper resulting from these studies was published in MBOC and three reviews were published Project 2: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Vinay Swaminathan Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization or contractility drive retrograde flow of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-V3 integrins in fibroblasts shows that integrins are co-aligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modelling, suggest that integrins in FA are co-aligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. This work resulted in two research publications that are in press at PNAS and Nat Comm.

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U.S. National Heart Lung and Blood Inst
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Luo, Ruibai; Chen, Pei-Wen; Kuo, Jean-Cheng et al. (2018) ARAP2 inhibits Akt independently of its effects on focal adhesions. Biol Cell 110:257-270
Wu, Zhanghan; Plotnikov, Sergey V; Moalim, Abdiwahab Y et al. (2017) Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing. Biophys J 112:780-794
Swaminathan, Vinay; Alushin, Gregory M; Waterman, Clare M (2017) Mechanosensation: A Catch Bond That Only Hooks One Way. Curr Biol 27:R1158-R1160
Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Mehta, Shalin B et al. (2017) Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc Natl Acad Sci U S A 114:10648-10653
Nordenfelt, Pontus; Moore, Travis I; Mehta, Shalin B et al. (2017) Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration. Nat Commun 8:2047
Zheng, Wei; Wu, Yicong; Winter, Peter et al. (2017) Adaptive optics improves multiphoton super-resolution imaging. Nat Methods 14:869-872
Thompson, Peter M; Ramachandran, Srinivas; Case, Lindsay B et al. (2017) A Structural Model for Vinculin Insertion into PIP2-Containing Membranes and the Effect of Insertion on Vinculin Activation and Localization. Structure 25:264-275
Baird, Michelle A; Billington, Neil; Wang, Aibing et al. (2017) Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells. Mol Biol Cell 28:240-251
Swaminathan, Vinay; Waterman, Clare M (2016) The molecular clutch model for mechanotransduction evolves. Nat Cell Biol 18:459-61
Skau, Colleen T; Fischer, Robert S; Gurel, Pinar et al. (2016) FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis. Cell 167:1571-1585.e18

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