Force production by actin polymerization motor at leading edge of locomoting cell

肌动蛋白细丝在片状脂体中被组织成网状物和束状物的亚室。肌动蛋白分子聚合成细丝，为细胞运动产生机械力。这些细丝的组织是通过肌动蛋白细丝结合蛋白来调节的。Fascin、LIM和SH3结构域蛋白1(LASP-1)和LASP-2在束上的定位表明它们参与了该组织；然而，它们的作用尚不清楚。我们比较了这些蛋白质和肌动蛋白在束处的周转情况。光漂白后，EGFP-肌动蛋白从束尖向内恢复，与踏车倒流一致。相反，EGFP-Fasin、-LASP-1和-LASP-2的恢复是从顺行方向发生的。这些结果表明，这些分子可能参与了束的稳定，但不参与启动。Lasp-2已被确定有三个结构域：LIM结构域、NeBulin-Repeat结构域和SH3结构域；然而，负责肌动蛋白结合的区域仍不清楚。我们已经在NG108-15和C2C12细胞中表达了EGFP标记的Lasp-2片段。我们发现，从LIM结构域到第一个星云蛋白重复模块的N-末端片段保持了肌动蛋白结合活性，并具有与全长LASP-2相似的亚细胞定位，但LIM结构域片段没有。LIM结构域的部分截断导致肌动蛋白结合活性的丧失和亚细胞定位的丧失。这些结果表明，在体内和体外，LASP-2与F-肌动蛋白的相互作用是通过LIM结构域和第一个NeBulin-Repeat模块的协同作用来实现的。

Actin filaments are organized into sub-compartments of meshwork and bundles in lamellipodia. Polymerization of actin molecules into filament produces a mechanical force for cell movement. Organization of these filaments is through to be regulated by actin filament binding proteins. Localization of fascin, the LIM and SH3 domain protein 1 (lasp-1), and lasp-2 to the bundles suggest their involvement in that organization; however, their contributions remain unclear. We have compared the turnover of these proteins with actin at the bundle. After photobleaching, EGFP-actin recovered inwards from the bundle tip, consistent with the retrograde flow by treadmilling. In contrast, the recovery of EGFP-fascin, -lasp-1 and -lasp-2 occurred from the anterograde direction. These results suggest that these molecules would participate in the stabilization of bundles but not in initiation.Lasp-2 has been identified to have three domains : a LIM domain, nebulin-repeat domain and an SH3 domain in lasp-2 ; however, the region responsible for actin-binding is still unclear. We have expressed lasp-2 fragments tagged with EGFP in NG108-15 and C2C12 cells. We showed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module retained actin-binding activity and a similar subcellular localization to full-length lasp-2, but the LIM domain fragment did not. Partial truncation of the LIM domain caused the loss of actin-binding activity and subcellular localization. These results suggest that lasp-2 interaction with F-actin is mediated by the cooperation of the LIM domain and first nebulin-repeat module both in vitro and in vivo.