682C Poster - 10. Cell biology: Cytoskeleton, organelles and trafficking
Saturday April 09, 1:30 PM - 3:30 PM

Authors:
Hemin Shah ¹; Alex Hoover ¹; Megan Gladwin ²; Trudi Schüpbach ²; Olivier Devergne ¹

Affiliations:
1) Department of Biological Sciences, Northern Illinois University, DeKalb, IL; 2) Department of Molecular Biology, Princeton University, Princeton, NJ

Keywords:
s. extracellular matrix; b. cell polarity

Epithelial cells play critical roles in the development and maintenance of an organism, and the establishment and maintenance of apical-basolateral polarity (ABP) are essential to their function and integrity. ABP is established, maintained, and tightly regulated via intracellular trafficking and environmental cues, such as those provided by the basement membrane (BM). The BM is a specialized sheet of extracellular matrix accumulating and underlying epithelial cells on their basal side. Despite the important roles of the BM in the architecture and functions of epithelial cells, little is known about the mechanism ensuring the exclusive basal restriction of the BM components. To study BM deposition, we use the follicular epithelium (FE) of the Drosophila ovary as our model system. The GEF/RabGTPase complex Crag/Rab10 is a key regulator of the biological pathway specifically dedicated to the basal restriction of BM components. However, the exact mechanism responsible for Crag’s polarized intracellular localization and its activity in BM polarity remains yet to be elucidated. In FE, it assumes a polarized localization and accumulates apically and laterally through yet unknown mechanisms. Importantly, specific localization of the GEF Crag is thought to be critical for the localized activation of Rab10. Thus, to understand how Crag controls the polarized deposition of BM proteins, it is critical to determine how Crag’s activity and localization are controlled. Crag is a multidomain protein containing the DENN domains at the N-terminus responsible for its GEF activity, a Calmodulin Binding Site (CBS) domain responsible for its calmodulin-binding activity, and a conserved C-terminus domain. To determine the domain(s) necessary for Crag’s localization and activity, we performed a structure-function analysis. Our data suggest that the CBS domain, but not the DENN domains, is important for the localization of Crag to the apical and lateral domains, suggesting a role of calmodulin in the subcellular localization of Crag. The DENN domains, however, are required, but not sufficient, to control the basal restriction of the BM components to the basal side of the epithelial cells, suggesting that the proper localization of Crag is required for its control of the polarized secretion of basement membrane proteins. Altogether, our data shed a light on the regulation of the activity and localization of Crag, a key component of the biological pathway that controls BM polarity.