Functional analysis of Rab27A and its effector Slp2-a in renal epithelial cells.

Polarized epithelial cells have two distinct plasma membrane domains, i.e., an apical membrane domain and a basolateral membrane domain, that are the result of polarized trafficking of proteins and lipids. Several members of the Rab-type small GTPases, which are general regulators of membrane trafficking, have been reported to be involved in the regulation of polarized trafficking in epithelial cells, but their precise role in polarized trafficking is poorly understood. In a recent study we used Madin-Darby canine kidney (MDCK) II cells as a model of polarized cells and concluded from the results that Rab27A and its effector synaptotagmin-like protein 2-a (Slp2-a) regulate apical transport of Rab27-bearing vesicles in polarized epithelial cells. Both Rab27A and Slp2-a are uniformly localized at the plasma membrane in subconfluent, non-polarized MDCK II cells, but their expression increases as the cells become polarized, and they are specifically localized at the apical membrane in polarized MDCK II cells (i.e., two-dimensional cell culture). Slp2-a is also localized at the apical membrane of tubular MDCK II cysts (i.e., three-dimensional cell culture) and promotes the formation of a single apical domain in the cysts by regulating polarized trafficking of Rab27-bearing vesicles. In this chapter we describe the assay procedures for analyzing the expression and localization of Rab27A and Slp2-a in non-polarized and polarized renal epithelial cells.