Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling
Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling
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DOI：
10.1242/jcs.03066
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发表时间：
2006-09-01
影响因子：
4
通讯作者：
Waterman-Storer, Clare M.
中科院分区：
文献类型：
--
作者：
Prigozhina, Natalie L.;Waterman-Storer, Clare M.
运动细胞表现出质膜蛋白从前沿向细胞中心的不断逆行流动，当与底物粘附耦合时，可能会推动细胞向前运动。在这里，我们的目的是验证一个假设，即在上皮细胞中，这些质膜成分通过极化的内/胞外循环传递，并且它们的正确循环是正常迁移所必需的。为此，我们在PtK1细胞中表达了编码gtp -限制性（S25N）和gtp -限制性（Q70L） Rab11b突变体的cDNA构建体，Rab11b是一种小的gtp酶，与循环后期有关，在此阶段内体循环室的膜成分被运输回质膜。令人惊讶的是，我们发现Rab11b突变体在小细胞岛随机迁移的PtK1细胞中的短暂表达导致细胞形态改变，实际上增加了细胞的运动速度。在单层伤口愈合实验中，两种突变蛋白的稳定表达也不会降低细胞迁移速度，而是影响迁移的方向性。我们还测试了与内吞循环有关的其他Rab蛋白的作用，并发现循环抑制程度与非定向细胞运动增加之间存在明确的相关性。
Locomoting cells exhibit a constant retrograde flow of plasma membrane proteins from the leading edge towards the cell center, which, when coupled to substrate adhesion, may drive forward cell movement. Here, we aimed to test the hypothesis that, in epithelial cells, these plasma membrane components are delivered via a polarized endo/exocytotic cycle, and that their correct recycling is required for normal migration. To this end, we expressed in PtK1 cells cDNA constructs encoding GDP-restricted (S25N) and GTP-restricted (Q70L) mutants of Rab11b, a small GTPase that has been implicated in the late stage of recycling, where membrane components from the endosomal recycling compartment are transported back to the plasma membrane. Surprisingly, we found that transient expression of the Rab11b mutants in randomly migrating PtK1 cells in small cell islands caused altered cell morphology and actually increased the velocity of cell locomotion. Stable expression of either mutant protein also did not decrease cell migration velocity, but instead affected the directionality of migration in monolayer wound healing assays. We have also tested the effects of other Rab proteins, implicated in endocytic recycling, and discovered a clear correlation between the degree of recycling inhibition and the increase in non-directional cell motility.