Identification and Characterization of Rab GTPases Involved in the Polarized Deposition of Basement Membrane Proteins in Epithelial Cells

Abstract

Epithelial tissue is composed of sheets of tightly adherent cells and is distributed throughout the body. The distinct apical-basal polarity exhibited by these cells is crucial to their ability to carry out various specialized functions. The basement membrane (BM), a specialized sheet of the extracellular matrix that directly contacts the basal side of the epithelia, aids in directing apicobasal polarity. Little is known about the specific mechanism by which BM proteins achieve polarized distribution in epithelial cells. Recently our lab identified Crag, a DENN domain protein that plays a specific role in the secretion of BM proteins. In the absence of Crag, BM proteins aberrantly accumulate on both the apical and basal surfaces of the epithelia. Because DENN domain proteins have been shown to have guanine exchange activity, it is possible that Crag interacts with Rab-GTPases in this process. Here, we perform a dominant-negative (DN) screen of the 31 Rab-GTPases that have been identified in Drosophila and evaluate the localization of the BM protein Viking in the follicular epithelium (FE). In our screen we identified two Rab GTPases, Rab8 and Rab10, involved in the polarized deposition of BM proteins. In follicle cells expressing Rab8DN and Rab10DN, we observed the aberrant accumulation of Viking on both the apical and basal sides of the epithelia. We found that in wild type follicle cells both Rab8 and Rab10 are localized to the basal side of the FE. Using RNAi knockdown, we confirmed that the loss of Rab10 results in the abnormal distribution of BM proteins to the apical surface, without disrupting the deposition of apical, basolateral, or junctional proteins. Lastly, in the absence of Rab10, BM proteins seem to be secreted outside the apical membrane of follicle cells. Taken together, these results demonstrate that Rab10 is specifically necessary for the polarized deposition of BM proteins to the basal side of the FE, and suggests that Rab10 and Crag act in the same pathway that ensures the polarized deposition of the BM proteins in epithelial cells.