The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

Abstract

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

Cingulin Regulates Claudin-2 Expression and Cell Proliferation through the Small GTPase RhoA

“…The paracellular flux assay was as described in Marzesco et al (2002). Briefly, MDCK cells were cultured in duplicate for 5 d to reach the steady state in 24-mm Transwells.…”

Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

“…Although this approach must be treated cautiously because the behavior of the transfected protein may be altered by overexpression and/or addition of GFP, it has proved useful in the study of numerous Rab proteins, including the closely related Rab8 (Ang et al, 2003), Rab13 (Marzesco et al, 2002), and yeast Sec4p (Schott et al, 2002). Although abnormal behaviors induced by overexpression cannot be excluded, our morphological and kinetic analyses demonstrated that cells expressing a GFP chimera of the wild-type Rab10 behaved very similarly to untransfected cells and that the effects of the different GFP-Rab10 constructs were consistent over a wide range of expression levels and were restricted to a subset of membrane transport pathways.…”

“…Like Sec4p, Rab8 and Rab13 have also been associated with polarized membrane transport. Rab8 has been found to regulate transport from the TGN to the basolateral membrane of polarized MDCK cells (Huber et al, 1993;Ang et al, 2003), and both Rab8 and Rab13 have been associated with junctional complex dynamics in epithelial cells (Zahraoui et al, 1994;Marzesco et al, 2002;Lau and Mruk, 2003;Kohler et al, 2004;Morimoto et al, 2005).…”

Re-engineering Primary Epithelial Cells from Rhesus Monkey Parotid Glands for Use in Developing an Artificial Salivary Gland

“…Claudin-1 is another key component of tight junctions typically detected along the lateral membranes between neighboring cells. 23 As shown in Fig. 1F (x-y and x-z images), incubating RPG cells with antibody to claudin-1 labeled the lateral membranes.…”