Department of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ, USA.
Macalester College, St. Paul, MN, USA.
Small GTPases. 2021 Jul;12(4):282-293. doi: 10.1080/21541248.2020.1754104. Epub 2020 Apr 17.
Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.
膜转运建立并维持上皮细胞极性。Rab22a 在激活的 T 细胞中呈极化分布，但它在上皮细胞极性中的作用尚未被研究过。我们之前曾表明，Rab14 在上游作用于 Arf6 以建立顶端膜起始位点（AMIS），但其与 Rab22a 的相互作用尚不清楚。在这里，我们显示 Rab14 和 Rab22a 在内质网体(endosomes)中在非极化和极化的 MDCK 细胞中共定位，并且 Rab22a 定位于极化细胞对的细胞:细胞界面。Rab22a 的敲低导致在三维培养中出现多腔表型。此外，在 Rab14 敲低细胞中过表达 Rab22a 可挽救 Rab14 敲低观察到的多腔表型，表明 Rab22a 是 Rab14 的下游。由于 Rab14 和 Arf6 之间的关系，我们研究了 Rab22a 敲低对 Arf6 的影响。我们发现 Rab22a 敲低导致活性 Arf6 减少，并且 Rab22a 与 Arf6 GEF EFA6 共免疫沉淀。此外，EFA6 在 Rab22a KD 细胞中保留在细胞内斑点内。这些结果表明，Rab22a 作为 Rab14 的下游，将 EFA6 转运到 AMIS 以调节 Arf6 在极性建立中的作用。
