Project 1: FMN2 makes perinuclear actin to protect nuclei during confined migration and promote metastasis. Colleen Skau Bob Fischer Hawa Thiam Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas, and show that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration, and thus promote cancer metastasis. This work was published in Cell Project 2:Mechanosensing by integrins regulates signaling and actin dynamics during phagocytosis Valentin Jaumouille Tissue resident phagocytes, such as macrophages and dendritic cells, act as sentinels of the immune system. They play a major role in the clearance of large particulate material, such as apoptotic cells and microbes. Depending on the nature of the particle they engulf, macrophages and dendritic cells will initiate an inflammatory response and present antigens to T lymphocytes. Phagocytosis depends on the reorganization of the actin cytoskeleton, driven by surface receptors. Although multiple signaling pathways have been identify, little is known about the molecular mechanisms underlying the formation of signaling complexes by the receptors, how actin reorganization is adjusted to the target biophysical properties, the mechanical forces involved in the uptake and whether they affect the downstream immune responses. We observed that engagement of 2 integrins by stiff particles lead to the assembly of a molecular platform that can act as a clutch to transduce mechanosensation. Whereas it has been previously established that 2-mediated phagocytosis of soft particles is independent of tyrosine kinases, we found that engulfment of stiff particles requires Src family kinases, Syk and the Arp2/3 complex. Live cell imaging reveals how target mechanical properties regulate actin dynamics in macrophages. This work was presented at two Gordon conferences and ASCB and several seminars in europe Project 3:Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells. Michelle Baird Bob Fischer The role of non-muscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, however whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A pulses occur in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or MLCK-mediated NM2 regulatory light chain phosphorylation, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. This work was published in Mol. Biol. Cell Project 4: YAP nuclear localization in the absence of cell-cell contact is mediated by a filamentous actin-dependent, myosin II- and phospho-YAP-independent pathway during ECM mechanosensing Arupratan Das YAP and TAZ transcriptional co-activators mediated up regulation of the target genes are responsible for development, tumor formation and cell differentiation. Nuclear exclusion of YAP through Hippo signaling mediated phosphorylation at S112 residue is well characterized. Actin being shown as common regulator of both Hippo signaling dependent as well as in Hippo independent regulation of YAP. Cellular perception of mechanical environment and regulation of YAP nuclear localization through actin is reported to determine differentiation fate. Here we showed that actomyosin contractility suppresses YAP phosphorylation at S112 residue however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. Essential player for YAP nuclear localization is F-actin, which even triggers pS112-YAP nuclear localization independent of myosin contractility. Such actin mediated regulation is also conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced F/G actin ratio leading to nuclear exclusion of both YAP and pS112-YAP. These data provide evidences for actomyosin contractility and phosphorylation independent regulation of YAP nuclear localization. In support to the physiological relevance, this study might help to explain reported observations where YAP dependent intestinal tissue growth persisted even in the activation of Hippo signaling and YAP phosphorylation at S112 residue This work was published in J. Biol. Chem. Project 5: Jeremy Logue and Richard Chadwick Abstract Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large leader bleb. Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement. This project resulted in 3 publications Project 6: Functional Behavior of Overexpressed Fusion Proteins in Melanoma Cells Under Confinement Mediating Leader Bleb Based Motility Greg Adams Cell movement is mediated by a remarkably plastic array of morphologies. Cancer cells migrating in confined 3D environments mimicking tissue microenvironment can switch between modes of migration depending on the degree of confinement and the availability of adhesive ligand. Under high confinement and low adhesion, cells undergo a mesenchymal-to-amoeboid switch and take on a highly stereotypical morphology with fast, persistent movement termed leader bleb-based migration (LBBM). The morphology is characterized by formation of a large ( 20.5 um) sausage-shaped bleb that points in the direction of movement (the leader bleb) separated from a smaller ( 12.6 um) sphe
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