Abstract
Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), we found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts.
Highlights
Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown
We found that the cytoplasmic tail of PODXL interacted selectively with Rab35WT and Rab35Q67L, but not with Rab35S22N, the GDP-bound, inactive form (Fig. 1a)
Endogenous PODXL could be co-immunoprecipitated from Madin-Darby canine kidney (MDCK) cells expressing Rab35WT or Rab35Q67L, but not from Rab35S22N, Rab6AQ72L, Rab8AQ67L, Rab11Q70L- or Rab27AQ78L-expressing cells (Fig. 1c)
Summary
Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Rab[35] physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site These vesicles transport aPKC, Cdc[42], Crumbs[3] and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab[35] and the cytoplasmic tail of Podocalyxin. We show that the Rab[35] GTPase is localized at the cell–cell interface and at the future AMIS during cytokinesis, where it captures vesicles transporting key apical determinants via a direct interaction with the cytoplasmic tail of PODXL Through this original mechanism of vesicle tethering, Rab[35] couples cell division with initiation of apico-basal polarity and lumen formation
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