Absence Of Myosin Light Chain Kinase Accelerates Smooth Muscle Cell Protrusion And Migration By Reducing Cell Membrane Tension

Cell migration, a continuous cycle that is spatiotemporally highly coordinated, plays a fundamental role in many important biological processes, such like angiogenesis, tissue repair, immune response and inflammation, cancer invasion and metastasis. A generally accepted model of cell migration includes several steps. Firstly, an initial cell polarization occurs via induction by migration-promoting or chemotactic agents in the extracellular environment, followed the formation of myosin activity involved actin-driven leading edge protrusion; then the attachment of the front of the cell to the extracellular matrix by assembly of integrin-mediated focal interactions; finally, disassembly of adhesions at the rear of the cell and the retraction of the cell’s tail by actomyosin-mediated contraction, which leads to the cell body translocates. The actomyosin-mediated contraction is dependent on the phosphorylation of the regulatory light chain (RLC) of myosin II. For many years, myosin light chain kinase (MLCK), a dedicated Ca2+/calmodulin-dependent protein kinase that phosphorylates the myosin regulatory light chain, has been proposed to play critical roles in cell migration, especially as a facilitative factor by generating essential contracting force. Published papers have suggested that blocking MLCK activity could impair cell migration. However, this conclusion was mainly based on the MLCK blocking experiments by chemical inhibitors with non-specificity. On account of so many influencing factors and spatiotemporally organized steps in cell migration, the exact role of MLCK is still a complex issue to be resolved.In this thesis, we established primary cultured MLCK-deficient (KO, knockout) mouse intestinal smooth muscle cells (SMCs) to investigate the alterations in cell morphology and migrating characteristics.To our surprise, MLCK-deficient SMCs started their outgrowth from the explants as early as at the4th cultured day, whereas most control (CTR) SMCs started to migrate out at the7th-9th day. Moreover, we observed that MLCK-deficient SMCs showed more enhanced lamellipodias and significant increased spreading and migrating speed comparing with control cells. Additionally, the MLCK-deficient SMCs displayed stronger protrusions formed at the leading edge as well as the faster protruding rate (average lamellipodial extension distance per30minute:KO:21.9±1.9μm vs. CTR:15.0±2.2μm, p<0.05). And MLCK-deficient SMCs showed larger spreading area at60min after cell inoculation (KO:20110±642μm2vs. CTR:8457±880μm2, p<0.0001). Quantification of wound healing assay showed a higher average migrated distance per hour in the MLCK-deficient SMCs (KO:55.3±2.5μm vs. CTR:19.3±1.7μm,p<0.001). Besides, there was an unexpected finding that the RLC phosphorylation was not significantly altered in primary cultured MLCK-deficient SMCs.In addition, mutant cells showed reduced cell membrane tension (by tether force measurement), with increased membrane motility and less cortical F-actin filaments. With rescue assay, we confirmed that both destabilized F-actin underneath plasma membrane and F-actin polymerization at the leading edge were essential for initiating protrusion formation. Furthermore, MLCK was found to have the binding activity to fibronectin-integrin-cytoskeleton complex by fibronectin pull-down assay. This result provide a preliminary molecular basis for that, MLCK can locate around the membrane and stabilize the cortical actomyosin cytoskeleton.In summary, our studies argue for a novel role for MLCK, which could be recruited by fibronectin-integrin-cytoskeleton complex, modifying cell protrusion and migration by controlling membrane tension through cortical F-actin stabilization. Thus, we have a conclusion that MLCK, serving as an F-actin bundling protein in a RLC phosphorylation independent way, maintains the plasma membrane tension at a high level before cell protruding. This finding sheds light on a novel membrane tension based mechanism underlying the regulation of cell migration. And it may describe a novel breakthrough point for therapeutics of cell migration-related diseases, such as neoplasm metastasis.