Prigozhina Natalie L, Waterman-Storer Clare M
Department Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, CB163, La Jolla, CA 92037, USA.
Curr Biol. 2004 Jan 20;14(2):88-98. doi: 10.1016/j.cub.2004.01.003.
Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.
To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.
These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.
运动细胞表现出质膜（PM）蛋白从前沿板状伪足向后持续逆行流动，当这种流动与底物黏附相结合时，可能驱动细胞向前移动。然而，这些质膜成分的细胞内来源以及它们的持续逆行流动是否是细胞运动所必需的尚不清楚。
为了验证顺行分泌途径为逆行流动提供质膜成分这一假设，而这种逆行流动是板状伪足活动和细胞运动所必需的，我们在瑞士3T3成纤维细胞中特异性抑制货物从反式高尔基体网络（TGN）向质膜的转运，并使用延时显微镜监测细胞运动。用蛋白激酶D1（PKD）的显性负性、激酶失活（kd）突变体抑制TGN到质膜的运输，该突变体特异性阻断分泌小泡从TGN出芽，且不影响其他运输途径。TGN上PKD的抑制抑制了细胞的定向运动以及表面标志物和丝状肌动蛋白的逆行流动，而细胞其他部位PKD的抑制既不阻断顺行膜运输也不阻断细胞运动功能。在表达PKD-kd的细胞中外源激活Rac1可恢复板状伪足动力学，且与膜运输无关。然而，板状伪足活动从单个前沿处发生了移位，细胞的定向运动并未完全恢复。
这些结果表明，PKD介导的从TGN到质膜的顺行膜运输是成纤维细胞运动和局部Rac1依赖性前沿活动所必需的。我们认为，极化分泌传递货物，这些货物指导局部信号传导，以实现定向迁移所必需的持续前沿活动。
