Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells.

The characteristic feature of polarity establishment in MDCK II cells is transcytosis of apical glycoprotein podocalyxin (PCX) from the outer plasma membrane to the newly formed apical domain. This transcytotic event consists of multiple steps, including internalization from the plasma membrane, transport through early endosomes and Rab11-positive recycling endosomes, and delivery to the apical membrane. These steps are known to be tightly coordinated by Rab small GTPases, which act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. However, our knowledge regarding which sets of Rabs regulate particular steps of PCX trafficking was rather limited. Recently, we have performed a comprehensive analysis of Rab GTPase engagement in the transcytotic pathway of PCX during polarity establishment in 2-dimensional (2D) and 3-dimensional (3D) MDCK II cell cultures. In this Commentary we summarize our findings and set them in the context of previous reports.

Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures.

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures.

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.