MEMBRANE TRAFFICKING AND THE ESTABLISHMENT OF POLARITY

Abstract

Differential membrane trafficking and modulation of lipid domains establishes and maintains cellular polarity in epithelial cells – these events are controlled largely by small GTPases. We have shown previously that Rab14 acts upstream of Arf6 in the establishment of the apical membrane, but how it interacts with other trafficking machinery is unknown. Rab22 has a polarized distribution in activated T-cells, but its role in epithelial polarity is not known. Here we report the colocalization of Rab14 with Rab22a in endosomes of Madin Darby canine kidney (MDCK) cells. Interestingly, Rab22 localizes to the cell:cell interface of polarizing cell pairs, and Rab14 and Rab22 colocalize in adjacent endosomes. Knockdown of Rab22 results in a multi-lumen phenotype in 3D culture, and overexpression of Rab22 in Rab14 knock down cells, results in the production of Rab22-positive extensions. Because of the relationship between Rab14, Rab22, and Arf6, we investigated the interaction of Rab22 with Arf6 GEFs and found that Rab22 co-immunoprecipitates with the Arf6 GEF EFA6. Furthermore, EFA6 is retained in intracellular puncta in Rab22 KD cells. These results suggest that Rab22 acts downstream of Rab14 to regulate Arf6 activity in the establishment of polarity.