Project 1: Rac1 induces PCK-dependent myosinIIA phosphorylation to regulate association with focal adhesions and cell migration. Pasapera AM1, Fischer RS1,Plotnikov SV1,Egelhoff T2, Waterman CM1. Cell Biology and Physiology Center, NHLBI, NIH1;Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic.2 Cell migration requires coordinated assembly of focal adhesions and contraction in the actomyosin cytoskeleton. The small GTPase Rac1 is critical to cell migration through its known functions in regulation of focal adhesion and actin cytoskeletal assembly dynamics, but its role in regulation of myosin II is not known. Myosin II dynamically assembles into minifilaments at the leading edge of migrating cells, and PKC-mediated phosphorylation in Ser 1916 in the non-helical tail is one of the main regulators. We hypothesized that Rac1 may regulate myosin II minifilament assembly dynamics during cell migration via downstream regulation of PKC and Ser 1916 phosphorylation. To test this, we analyzed the effects of Rac1 activation on the phosphorylation and dynamics of myosin IIA in U2OS cells. We found that transfection of active Rac1 (Rac1V12) induced PKC- and integrin-dependent myosin IIA phosphoryation on Ser 1916. Live cell imaging of GFP-myosin IIA revealed that Rac1 activation promotes rapid assembly, motion, and turnover of myosin IIA minifilaments, as well as perpendicular orientation to the leading edge, resulting in its accumulation specifically in focal adhesions. To determine the role of Ser 1916 phosphorylation on myosin IIA dynamics and localization, we expressed phospho-mimetic (S1916D) and non-phosphorylatable mutants (S1916A) of myosin IIA. This showed that phosphorylation is critical to the Rac1-induced rapid assembly and turnover of myosin IIA minifilaments as well as to the focal adhesion association of myosin IIA. Thus, Rac1 acts as a master regulator of cell migration by coordinating actin assembly-mediated protrusion, adhesion, and actomyosin contraction dynamics. This work was published in Current Biology Project 2:Mechanosensing by 2integrins regulates signaling and actin dynamics during phagocytosis Valentin Jaumouill 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 will be presented at a Gordon conference and ASCB Project 3: Analysis of traction stress variation across single focal adhesions. Sergey V. Plotnikov Using high-resolution traction force microscopy on polyacrylamide ECMs of varying stiffnesses, we found that the mechanical behavior of the integrin-actin interface at FA exhibited ECM stiffness-dependent switching between a load-and-fail compliance sensing regime and a frictional slippage regime as described in the clutch oscillation model (Chan and Odde, 2008). In the load and fail regime, the position of peak traction within the FA resided on average at the distal FA tip, but oscillated over time towards the FA center and back to the tip. As ECM rigidity was increased, the traction peak did not oscillate and remained in the FA center, signifying the frictional slippage regime. We found that perturbing the gradient of paxillin phosphorylation across FA by expressing Y31/118E- or Y31/118F-paxillin mutants or by inhibiting FAK weakened the molecular clutch and switched FAs from load-and-fail compliance sensing to frictional slippage regime on compliant ECMs. In agreement with the clutch oscillation model, the load-and-fail regime could be rescued by further decreasing either substrate stiffness or myosin II contractility. Since paxillin phosphorylation on tyrosine residues 31 and 118 mediates vinculin recruitment into FAs, we demonstrated that vinculin and the paxillin-vinculin interaction are essential to strengthen the molecular clutch and to enable mechanosensing over a wide range of ECM compliances. We demonstrated the physiological importance of the load-and-fail compliance sensing regime by showing a requirement for this FA behavior in durotaxis, but not in chemotaxis in a boyden chamber assay or in random cell migration. This work resulted in 2 publications 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 has been submitted to the J. Biol. Chem.
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