Chen Pin-I, Schauer Kristine, Kong Chen, Harding Andrew R, Goud Bruno, Stahl Philip D
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
Molecular Mechanisms of Intracellular Transport, Institut Curie, Paris, France.
PLoS One. 2014 Feb 28;9(2):e90384. doi: 10.1371/journal.pone.0090384. eCollection 2014.
Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.
Rab5是内吞途径的典型Rab GTP酶和主要调节因子，它编码为三种差异表达的异构体，即Rab5A、Rab5B和Rab5C。在此，我们研究了Rab5异构体沉默对细胞运动的不同影响，并报告Rab5C，而非Rab5A或Rab5B，与Rac1的生长因子激活以及增强的细胞运动选择性相关。初步观察表明，Rab5C表达的沉默，而非Rab5A或Rab5B的沉默，导致细胞呈纺锤形，膜皱褶形成减少。在划痕实验中，Rab5C缺失的细胞，而非Rab5A或Rab5B缺失的细胞，显示出细胞迁移减少。在Transwell板迁移实验中，Rab5C缺失的U937细胞也表现出细胞运动减少。为了检测Rac的激活，稳定表达GFP-Rac1的HeLa细胞分别缺失Rab5A、Rab5B或Rab5C，然后接种到印有弩形图案的盖玻片上。微图案化细胞的3D GFP-Rac1图像显示，在没有Rab5C的情况下，GFP-Rac1较少定位于细胞周边。为了证实Rab5C与Rac激活之间的联系，缺失Rab5异构体的HeLa细胞饥饿后用表皮生长因子（EGF）刺激。Rac1下拉实验表明，在Rab5C抑制的细胞中，EGF刺激的Rac1活性显著受到抑制。为了确定Rac激活上游的事件是否受Rab5C影响，我们观察到在缺失Rab5C的细胞中，EGF刺激的Akt磷酸化受到抑制。最后，由于黏附复合物的时空组装/拆卸是细胞迁移的重要组成部分，我们研究了Rab5异构体缺失对粘着斑复合物形成的影响。与缺乏持续的片状伪足突起和定向迁移减少一致，Rab5C缺失的HeLa细胞粘着斑显著减少。我们得出结论，Rab5异构体选择性地监督与内吞网络相关的多个信号和运输事件。
