Cell-surface biotinylation to study endocytosis and recycling of occludin.

The dynamic turnover of adherens junctions (AJs) and tight junctions (TJs) is essential for epithelial morphogenesis during normal development and differentiation. Although the endocytic recycling of E-cadherin is characterized and implicated in AJ turnover, the molecular basis for TJ turnover is poorly understood. Occludin and claudins are distinct transmembrane proteins localized to the TJs. Although claudins are an indispensable structural component of TJ strands, depletion of occludin in mice reveals well-developed TJ strands and complex histological abnormalities. To examine the intracellular transport of transmembrane proteins to and from the cell surface, cell-surface biotinylation is a proven powerful method. Using this method, we successfully demonstrated that occludin was endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney (BHK) and epithelial MTD-1A cells. The endocytic recycling of occludin as well as the formation of functional TJs was dependent on Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2). We describe the method to study the intracellular transport of occludin to and from the cell surface in both fibroblastic and epithelial cells.