Abstract
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.
Highlights
Migrating cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backwards towards the cell center
In our previous work (Prigozhina and Waterman-Storer, 2004) we showed by inhibiting the budding of membrane cargo from the trans-Golgi network using a dominant negative mutant of protein kinase D that, in fibroblasts, directional locomotion depends on the anterograde secretion pathway
Localization of Rab11b in PtK1 cells We aimed to test for the requirement of the slow endosomal recycling pathway in PtK1 epithelial cell motility by disrupting the activity of Rab11b
Summary
Migrating cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backwards towards the cell center. Two possible mechanisms for supplying these materials to the leading edge of migrating cells have been proposed. One suggests that the components may be delivered via a polarized endo/exocytotic cycle (i.e. recycling) (Bretscher, 1992; Bretscher, 1984; Bretscher and AguadoVelasco, 1998b; Hopkins et al, 1994), and the other suggests that they maybe newly synthesized and delivered to the leading edge via the anterograde secretion pathway (Bergmann et al, 1983; Prigozhina and Waterman-Storer, 2004). It is possible that migrating fibroblasts and epithelial cells may preferentially rely on different membrane trafficking pathways to supply PM components for retrograde flow and leading edge advancement
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