Mettlen Marcel, Platek Anna, Van Der Smissen Patrick, Carpentier Sarah, Amyere Mustapha, Lanzetti Letizia, de Diesbach Philippe, Tyteca Donatienne, Courtoy Pierre J
CELL Unit, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology, 1200 Brussels, Belgium.
Traffic. 2006 May;7(5):589-603. doi: 10.1111/j.1600-0854.2006.00412.x.
We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.
我们研究了Src在携带温度敏感型v-src突变体的MDCK细胞中对皮质肌动蛋白动力学和极化内吞作用的作用。将在40℃(非允许温度)建立的单层细胞转移到34℃(允许温度)会迅速重新激活v-Src激酶,但紧密连接和细胞极性在>6小时内仍能维持。在此期间,活化的v-src被募集到顶端小泡上,诱导与cortactin相关的顶端圆形褶皱,产生巨胞饮体,从而使顶端胞饮作用加速约五倍。肌动蛋白聚合、磷酸肌醇3激酶、磷脂酶C和磷脂酶D的抑制剂可选择性地消除褶皱和巨胞饮体形成,这些抑制剂均使顶端胞饮作用恢复到40℃时观察到的水平,突出了顶端微胞饮作用和巨胞饮作用的不同调控方式。Src促进微管依赖性巨胞饮体与顶端回收内体(ARE)融合,导致其强烈空泡化。然而,微管形成和顶端极性的保留表明其功能未受影响。ARE被标记为v-src、Rab11和rabankyrin-5,但未被早期内体抗原1标记,从而区分了两条独立的Rab-5依赖性顶端途径。Src诱导的顶端褶皱和巨胞饮作用机制可能有助于阐明引发的顶端肠道侵袭性病原体的进入以及破骨细胞的顶端分化。