Pasapera Ana M, Plotnikov Sergey V, Fischer Robert S, Case Lindsay B, Egelhoff Thomas T, Waterman Clare M
Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA; Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Curr Biol. 2015 Jan 19;25(2):175-186. doi: 10.1016/j.cub.2014.11.043. Epub 2014 Dec 24.
Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear.
We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness.
Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
细胞迁移需要粘着斑（FAs）的协调形成以及肌动蛋白细胞骨架的组装和收缩。非肌肉肌球蛋白II（MII）是细胞迁移中收缩性和粘着斑动态变化的关键调节因子。小GTP酶Rac1下游的信号传导也调节粘着斑和肌动蛋白动态变化，但其在迁移过程中对MII调节的作用尚不清楚。
我们发现Rac1促进MIIA与粘着斑的结合。活细胞成像显示，虽然前缘的大多数MIIA组装成背侧收缩弧，但相当一部分在成熟的粘着斑内组装或被捕获，并且这种行为由活性Rac1促进。蛋白激酶C（PKC）激活对于MIIA重链（HC）丝氨酸1916（S1916）上的整合素和Rac1依赖性磷酸化以及募集到粘着斑是必要且充分的。在细胞铺展和细胞外基质硬度机械传感过程中，MIIA HC的S1916磷酸化和在粘着斑中的定位增强，表明在生理性Rac1激活过程中该途径上调。磷酸模拟和不可磷酸化的MIIA HC突变体表明，S1916磷酸化对于MIIA微丝在粘着斑中的捕获和组装是必要且充分的。S1916磷酸化也足以促进粘着斑的快速组装以增强细胞迁移，并通过细胞外基质硬度调节牵引力、铺展和迁移。
我们的研究首次揭示，Rac1和整合素激活通过PKC依赖性机制调节MIIA HC磷酸化，该机制促进MIIA与粘着斑的结合，并作为细胞迁移和机械传感的关键调节因子。
