Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability.
Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.
Benais-Pont
Gaelle
G
Department of Cell Biology, University of Geneva, 1211 Geneva, Switzerland.
Punn
Anu
A
Flores-Maldonado
Catalina
C
Eckert
Judith
J
Raposo
Graca
G
Fleming
Tom P
TP
Cereijido
Marcelino
M
Balda
Maria S
MS
Matter
Karl
K
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002450:Cell Communication / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000235:genetics; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification*; D034622:RNA Interference / Q000502:physiology; D064067:Rho Guanine Nucleotide Exchange Factors; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D019108:Tight Junctions / Q000378:metabolism* / Q000648:ultrastructure; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
C577356:ARHGEF2 protein, human; C577402:Arhgef2 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins