A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells

Abstract

Abstract. Small rab/Yptl/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rabl3, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rabl3 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rabl3 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca 2+ media induces the redistribution of rabl3. In cells devoid of tight junctions, rabl3 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rabl3 to the resulting adherens-like junction complexes. The participation of rabl3 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.UCARYOTIC cells contain a variety of small 21-27 kd guanine nucleotide-binding proteins, in addition to H-, K-, and N-p21ras. Although these proteins are structurally distinct from each other and from p21ras, they share significant homologies with p21ras, particularly in the domains involved in GTP/GDP binding and in GTP hydrolysis. Numerous members of the ras-related GTPase superfamily have been identified and classified according to their sequence similarities (for review see Valencia et al., 1991). Among them, the proteins of the rab/Ypt/Sec4 family (>30 members) have been proposed as key regulators in vesicular traffic (Touchot et al

Rab13-dependent Trafficking of RhoA Is Required for Directional Migration and Angiogenesis

“…This is in agreement with previous studies reporting that in unpolarized cells, such as the ECs shown in Fig. 1 (D-G), Rab13 is present exclusively in cytoplasmic vesicles (33). When placed under a VEGF-A 164 gradient (0 -20 ng/ml) in a Dunn chamber, ECs formed lamellipodia and polarized in the gradient direction (Fig.…”

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

“…Similarly to GFP-Rab13 constructs, we found that VSV-Rab13Q67L localized to the plasma membrane and VSV-Rab13T22N to cytoplasmic structures (supplementary material). Moreover, we also examined the distribution of an other GFP-tagged protein Rab8, which shares high amino acid identity with Rab13 (Zahraoui et al, 1994) and is also associated with tight junctions in MDCK cells (Huber et al, 1993). GFP-Rab8Q67L localized to perinuclear region and to the plasma membrane (supplementary material) was consistent with the localization of endogenous Rab8 in MDCK cells (Huber et al, 1993).…”

“…The transepithelial electrical resistance (TER) can be used to monitor the tightness of the seal between neighboring cells. Because the recruitment of Rab13 to the apicolateral junctional complex occurred at an early stage in the assembly of TJ (Zahraoui et al, 1994;Sheth et al, 2000), we postulated that it might be involved in TJ sealing. Therefore, we measured TER over a 120-h time period of parental MDCK, GFP, GFP-Rab13WT, GFP-Rab13T22N, and GFP-Rab13Q67L cells grown on filters (Figure 2).…”

“…In MDCK cells, antibodies against Sec8, an exocyst subunit, inhibit vesicles delivery to the basolateral membrane (Grindstaff et al, 1998). Three small GTPases, Rab3b, Rab8, and Rab13, are localized in cell-cell contacts in epithelial cells (Huber et al, 1993;Weber et al, 1994;Zahraoui et al 1994). Rab proteins are involved in the regulation of different steps of exocytic and endocytic pathways (Schimmoller et al, 1998;Chavrier and Goud, 1999;Zerial and McBride, 2001).…”

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

“…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).…”