Integration of Actin Dynamics and Adhesion in Cell Migration

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Project: MARK2/Par1b regulates cytoskeleton polarization and directed cell migration through modulation of Myosin II contractility and focal adhesion organization. Personnel: Ana Pasapera Collaborators: Sarah Heissler, Masumi Eto Goal: We sought to test the hypothesis that MARK2 mediates polarization and directed migration of cancer cells through regulation of the actomyosin cytoskeleton. In this work we uncovered new roles for the MT regulator and polarity protein MARK2 in actomyosin contractility and FA organization in migrating human cancer cells. Our analyses in osteosarcoma cells showed that in addition to the MT cytoskeleton and plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and FAs. Together, our results define MARK2 as a master regulator of the spatial organization of the actomyosin and MT cytoskeletal systems and FAs to mediate polarization and steering of cancer cell migration. Project: Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of Rock2. Personnel: Jessica Wayt Collaborators: Alex Cartegena-Rivera Goal: Determine the mechanism by which Rho-associated kinases (Rocks) mediate CIE. We discovered that ROCK2-mediated myosin II contractility promotes CIE, and that different CIE cargos in different cellular domains utilize distinct myosin II isoforms. Together, this study demonstrates that specific CIE cargoes are internalized by ROCK2-mediated activation of myosin II isoforms in distinct cell regions, possibly by modulation of local cortical tension. Project: CDK-1-mediated phase dissolution of Tensin1 condensates contributes to focal adhesion disassembly at mitosis to relieve an integrin inactivation G2-M checkpoint. Personnel: Martina Lerche Collaborators: Jeffrey Urbach Goal: Test the hypothesis that mitotic cell rounding is mediated by reducing cell-ECM adhesion through timely regulation of focal adhesion (FA) proteins by mitotic promoting factors. We found that cell de-adhesion is an early prophase event that occurs perfectly coincidentally with mitotic cell rounding and cyclin B nuclear translocation, and is accompanied by a rapid drop in traction forces with no apparent pre-de-adhesion peak. Taken together, our data suggest that the mitotic-promoting factor CDK1 regulates cell de-adhesion at mitosis by mediating massive phosphorylation of the integrin-actin linker, tensin 1 in its IDR. This phosphorylation potentially drives the dissolution of tensin 1-containing biomolecular condensates and tensin 1 early removal from FA, leading to integrin inactivation and FA disassembly. Our data further suggest that cell de-adhesion at mitosis is a checkpoint required for cell cycle progression in adherent cells. We are right this very second making non-phosphorylatable and phosphomimetic tensin 1 variants with all the Ss and Ts in the IDR mutated to see if the phosphorylation is required for dissolution of the droplets, and if dissolution of droplets is required for FA disassembly at mitosis and for cell cycle progression. Project: Role of ezrin in ECM stiffness sensing. Zbyszek Baster is working on the role of ezrin, radixin and moesin family (ERM) proteins in cell mechnosensation. ERMs are the main proteins linking the actin cortex to the cell plasma membrane118. ERM protein activity is regulated through their N-terminal binding to PIP2 rich plasma membranes followed by their phosphorylation at T567/T564/T558 (ezrin/radixin/moesin, respectively) by the homologous kinases, LOK or SLK, or by the Rho/ROCK pathway, to promote binding of the ERM C-terminal domains to actin119121. Activation of ERMs thus drives cortex-membrane tethering, which serves as one means of increasing cell stiffness and surface tension122. Since it is well established that mechanosensing of stiff ECM causes cell cortical stiffening89,122,123, it seems sort of shocking that little is known about whether ERM proteins are regulated in response to different mechanical stimuli. Zbyszek will thus test the hypothesis that cells respond to mechanical parameters of their environment in an ERM-dependent manner, and will dissect the signaling pathway mediating that response. He will use polyacrylamide or silicone cell culture substrates of a defined Young modulus and the Strex cell stretcher we plan to buy. ERM expression, activation and distribution will be analyzed in fibroblasts using molecular biology- and light microscopy-based techniques. ERM proteins and known ERM regulation or mechanotransduction pathways will be inhibited or stimulated using drug and gene expression manipulations to find molecular mechanisms by which ERM mechanosensation and mechanoresponse are mediated. Cell mechanical properties will be measured by AFM. This study will elucidate the mechanism of stiffness-dependent control of cell mechanical properties.