Vinculin stabilizes nascent adhesions and establishes lamellipodium-lamella border in migrating cells Ingo Thievessen, R. Ross The actin cytoskeleton at the leading edge of migrating cells consists of two actin networks, the lamellipodium (LP), characterized by fast polymerization-driven retrograde actin flow and the lamellum (LM) with slow myosin II (myoII) mediated actin flow. The engagement of LP actin to the ECM via nascent integrin-mediated focal adhesions (FA) establishes the flow velocity gradient between LP and LM. Nascent adhesions then elongate and mature via myoII LM actin flow. How integrins are connected to the retrograde actin flow is not known. Using primary murine embryonic fibroblasts (MEF) deficient for the vinculin gene (Vcl), we sought to test the hypothesis that vinculin mediates the coupling of actin retrograde flow to the ECM in FA . Single Vcl-/- MEF migrated faster than control (Vclfl/fl) MEF and displayed impaired anisotropic spreading. To determine if LP/LM organization were effected by vinculin deletion, we analyzed distributions of phospho-myosin light chain (pMLC), cortactin, and paxillin. This revealed a shift in pMLC distribution towards the cell edge, reduced LP paxillin intensity, and broadening of the contactin band at the leading edge. This suggests loss of delineation between the LP and LM. To test this, we performed spinning disc confocal (SDC) microscopy of MEF containing fluorescent paxillin and actin. This revealed reduction in the rate of formation of short-lived, diffraction limited FA in the LP of Vcl-/- MEF, indicating an impaired stabilization of nascent LP FA. Kymograph analyses of high resolution DIC and quantitative fluorescent speckle SDC microscopy of actin indicate the lack of two distinct velocity zones of retrograde f-actin flow near the leading edge and an increased retrograde flow velocity in the LM region of Vcl-/- MEF. We suggest that vinculin stabilizes nascent FA by coupling to lamellipodial actin flow, thus establishing the flow velocity gradient between LP and LM and promoting the maturation of nascent adhesions. This implicates vinculin as an essential component in linking the dynamic actin cytoskeleton to the ECM during cell migration. This work was done in collaboration with Bob Ross, and has been presented at Cell Biology Society meeting and Gordon Conference. Project 2: Nanoscale architecture of integrin-based cell adhesions Pakorn Kanchanawong Focal adhesions (FAs) mediate cell interactions with their extracellular matrices (ECMs) and consist of integrin ECM receptors linked to the actin cytoskeleton via plasma-membrane-associated protein plaques. Despite their fundamental importance in multicellular organisms, the three-dimensional organization of proteins within FAs is unknown. Here we determine FA molecular architecture by using 3D superresolution microscopy (interferometric Photo-Activated Localization Microscopy) to map nanoscale protein organization. We find that the FAs consist of partially overlapping proteinspecific vertical layers of 15-50 nm thickness, with integrins and actin separated by a 30- 50 nm FA core which is spanned by talin tethers. This reveals a structural basis for FA function whereby a multilaminar core architecture mediates the interdependent cell processes of adhesion, signaling, force transduction, and actin cytoskeletal regulation. This work was done in collaboration with Mike Davidson, Gleb Stengle, Harald Hess and has been revised and resubmitted at Nature. Project 3: Actin dynamics correlate with force transmission through focal adhesions Clare Waterman How focal adhesions (FAs) convert retrograde actin filament (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined Traction Force and Fluorescent Speckle Microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family GTPase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration. This work was performed in collaboration with Margaret Gardel (University of Chicago), Gaudenz Danuser and Lin Ji(Scripps), Ulrich Schwartz and Benedikt Sabass (Heidelberg University) and was accepted for publication. Project 4:A zyxin-mediated mechanism for actin stress fiber maintenance and repair. Clare Waterman
| 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 |
| 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 |
| 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; 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 |
Showing the most recent 10 out of 32 publications
