Luo Weiwei, Lieu Zi Zhao, Manser Ed, Bershadsky Alexander D, Sheetz Michael P
Mechanobiology Institute, National University of Singapore, Singapore, 117411, Singapore.
sGSK Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Proteos Building, 61 Biopolis Drive, Singapore, 138673, Singapore.
PLoS One. 2016 Oct 19;11(10):e0163915. doi: 10.1371/journal.pone.0163915. eCollection 2016.
A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.
在没有应力纤维的情况下，节点细胞质肌动蛋白网络是肌动蛋白细胞质凝聚的基础。我们之前描述过这样一个在Latrunculin A（LatA）处理后形成的网络，其中formin DAAM1定位于这些节点。敲低DAAM1会降低肌动蛋白节点的移动性，但节点仍然存在。在这里，我们研究了LatA洗脱后含DAAM1的节点。发现DAAM1分布在细胞质和质膜之间。膜结合可能通过与脂筏的相互作用发生，但不是F-肌动蛋白组装所必需的。有趣的是，通过雷帕霉素依赖性连接使DAAM1与质膜发生强制相互作用，在LatA洗脱后增强了细胞膜（与细胞质相比）处的F-肌动蛋白组装。然而，在同时添加雷帕霉素和LatA后立即观察到，在DAAM1移动到膜之前，细胞质肌动蛋白节点会短暂形成。这与DAAM1最初锚定在细胞质肌动蛋白节点的观点一致。此外，对DAAM1的光激活追踪显示DAAM1固定在这些肌动蛋白节点上。因此，我们认为DAAM1将肌动蛋白丝组织成节点复合体，并且这种节点复合体在肌动蛋白解聚后启动肌动蛋白网络的恢复。
