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

肌动蛋白丝被组织成板状伪足中的网状结构和束。肌动蛋白分子聚合成丝产生细胞运动的机械力。这些丝的组织是通过肌动蛋白丝结合蛋白来调节的。肌成束蛋白、LIM 和 SH3 结构域蛋白 1 (lasp-1) 和 lasp-2 定位于纤维束，表明它们参与该组织；然而，他们的贡献仍不清楚。我们将这些蛋白质与束处肌动蛋白的周转率进行了比较。光漂白后，EGFP-肌动蛋白从束尖向内恢复，与跑步机的逆行流一致。相反，EGFP-肌成束蛋白、-lasp-1和-lasp-2的恢复发生在顺行方向。这些结果表明这些分子将参与束的稳定，但不参与启动。Lasp-2 已被鉴定具有三个结构域：lasp-2 中的 LIM 结构域、nebulin 重复结构域和 SH3 结构域；然而，负责肌动蛋白结合的区域仍不清楚。我们在 NG108-15 和 C2C12 细胞中表达了带有 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 端片段保留了肌动蛋白结合活性以及与全长 lasp-2 类似的亚细胞定位，但 LIM 结构域片段却没有。 LIM 结构域的部分截断导致肌动蛋白结合活性和亚细胞定位的丧失。这些结果表明，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 重复模块在体外和体内的协作介导的。

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.