The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells.
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.
Marzesco
Anne-Marie
AM
Laboratory of Morphogenesis and Cell Signaling, UMR144 Centre National de la Recherche Scientifique (CNRS), Institut Curie, Paris, France.
Dunia
Irene
I
Pandjaitan
Rudy
R
Recouvreur
Michel
M
Dauzonne
Daniel
D
Benedetti
Ennio Lucio
EL
Louvard
Daniel
D
Zahraoui
Ahmed
A
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology* / Q000648:ultrastructure; D005614:Freeze Fracturing; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins