The urinary bladder is lined by a stratified (urothelium) that protects the underlying tissues from the non-physiological urine composition by forming a differentiated umbrella cell layer that elaborates an apical plasma membrane with a crystalline, asymmetric unit membrane (AUM) structure. This unique membrane is mainly composed of four polypeptides, the uroplakins (UPs) Ia, Ib, II and III, which assemble in post-Golgi vesicles (discoidal and fusiform vesicles) into two-dimensional crystalline arrays. Fusiform vesicles function in the regulated insertion of pre-assembled AUMs into the apical membrane of umbrella cells. Thus, the urothelium offers unique opportunities to study the processing, assembly and targeting of membrane proteins in polarized epithelial cells. In our efforts to understand the structural requirements for uroplakin assembly we will express in 293T cells modified forms of UPs to monitor the formation of specific UP pairs. Using MDCK zells as a polarized epithelial cell model, we will test whether the information for apical targeting of the UPIb/UPIII heterodimer is contained in the cytoplasmic domain of UPIII. We have demonstrated that Rab27b is rather selectively expressed in urothelial cells where it binds to fusiform vesicles. To test our working hypothesis that Rab27b functions in the delivery of fusiform vesicles to the apical plasma membrane, we plan to identify and characterize homologues of melanophilin and other not yet identified proteins that interact directly or indirectly with Rab27b. We will also generate conditional knockout mice where Rab27b expression is eliminated in an urotheliun-specific fashion. To this effect we will cross mice that express cre under the control of the UPII promotor with a strain that harbors a """"""""fluxed"""""""" form of Rab27b. The morphological, functional and physiological characterization of these knockout mice compared with wild-type mice is expected to provide information on the specific step(s) in which Rab27b functions in the delivery of fusiform vesicles to the apical plasma membranes of urothelial umbrella cells. Such studies should help us better understand the normal physiology of the urothelium, and the mechanisms involved in bladder infection and bladder cancer.
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