Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.
Apical lumen formation is a key step during epithelial morphogenesis of tubular organs. Appropriate transport and targeting of apical proteins to the apical membrane initiation site (AMIS) plays a crucial role in establishing a solitary, central lumen. FIP5, a Rab11-interacting protein, is an important regulator that directs apical endosome trafficking along microtubules toward the AMIS during cytokinesis. However, it is unknown which molecular motor(s) transports FIP5-positive apical endosomes during lumen initiation, and how this process is regulated. In this study, we demonstrate that the interaction of FIP5 with the microtubule motor, Kinesin-2, is required for the movement of FIP5-endosomes and delivery of these endosomes from centrosomes to the cleavage furrow during apical lumen initiation. Loss of Kinesin-2 disrupts targeting of apical proteins to the AMIS and results in multiple lumen formation in MDCK cysts. Our data provide more details to the molecular mechanism of FIP5-dependent apical trafficking during apical lumen formation.
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