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TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

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Localization of TBC1D9B at sites of active Rab11a function. (A) Filter-grown MDCK cells were pulse labeled with 200 μg/ml IgA at the basolateral surface for 20 min at 18°C and chased for 20 min at 37°C in the presence of apical Cy5-labeled anti-IgA antibodies. The cells were fixed and then immunolabeled to detect endogenous TBC1D9B (green), endogenous Rab11a (red), and transcytosed IgA (blue). In the merged panel, the boxed region is magnified in the bottom right inset. Magnified views of the individual channels are found in the insets under ZOOM. (B, C) MDCK cells were stained for endogenous TBC1D9B and either the (B) pIgR or (C) endogenous Sec15A. (D) Coefficient of colocalization for endogenous TBC1D9B and the indicated proteins. (E, F) MDCK cells were transfected with cDNA encoding GFP-Rab11a-WT (E) or GFP-Rab11a-QL (F) and after 72 h fixed and then stained for endogenous TBC1D9B. (G) Coefficient of colocalization for TBC1D9B and the indicated Rab11a construct. In (B, C, E, and F), the boxed region is magnified in the panels under the label ZOOM. Scale bar, 5 μm. In D and G, data were obtained from at least three independent experiments, and the means ± SEM are shown (n ≥ 150 cells). In G, a Student's t test was used to show that difference between these two sample groups was significantly different (*p < 0.05).
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Figure 7: Localization of TBC1D9B at sites of active Rab11a function. (A) Filter-grown MDCK cells were pulse labeled with 200 μg/ml IgA at the basolateral surface for 20 min at 18°C and chased for 20 min at 37°C in the presence of apical Cy5-labeled anti-IgA antibodies. The cells were fixed and then immunolabeled to detect endogenous TBC1D9B (green), endogenous Rab11a (red), and transcytosed IgA (blue). In the merged panel, the boxed region is magnified in the bottom right inset. Magnified views of the individual channels are found in the insets under ZOOM. (B, C) MDCK cells were stained for endogenous TBC1D9B and either the (B) pIgR or (C) endogenous Sec15A. (D) Coefficient of colocalization for endogenous TBC1D9B and the indicated proteins. (E, F) MDCK cells were transfected with cDNA encoding GFP-Rab11a-WT (E) or GFP-Rab11a-QL (F) and after 72 h fixed and then stained for endogenous TBC1D9B. (G) Coefficient of colocalization for TBC1D9B and the indicated Rab11a construct. In (B, C, E, and F), the boxed region is magnified in the panels under the label ZOOM. Scale bar, 5 μm. In D and G, data were obtained from at least three independent experiments, and the means ± SEM are shown (n ≥ 150 cells). In G, a Student's t test was used to show that difference between these two sample groups was significantly different (*p < 0.05).

Mentions: Next, we determined whether TBC1D9B was localized to vesicle populations where Rab11a was likely to be in its active, GTP-bound state. One such vesicle pool is the apical recycling endosome, which is accessed by the pIgR late in its transit from the basolateral-to-apical poles of the cell (Apodaca et al., 1994; Casanova et al., 1999; Wang et al., 2000a). As predicted, endogenous TBC1D9B colocalized with Rab11a and the basolaterally internalized pIgR ligand, IgA, in subapical vesicles (coefficient of colocalization of 0.59 ± 0.02; Figure 7, A and D). In addition, TBC1D9B colocalized with vesicles positive for the pIgR (coefficient of colocalization of 0.54 ± 0.07; Figure 7, B and D) or the Rab11a (and Rab8a) effector, Sec15A, one subunit of the octameric exocyst complex (coefficient of colocalization of 0.33 ± 0.04; Figure 7, C and D; Guo et al., 1999; Zhang et al., 2004; Wu et al., 2005; Novick et al., 2006; Oztan et al., 2007).


TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

Localization of TBC1D9B at sites of active Rab11a function. (A) Filter-grown MDCK cells were pulse labeled with 200 μg/ml IgA at the basolateral surface for 20 min at 18°C and chased for 20 min at 37°C in the presence of apical Cy5-labeled anti-IgA antibodies. The cells were fixed and then immunolabeled to detect endogenous TBC1D9B (green), endogenous Rab11a (red), and transcytosed IgA (blue). In the merged panel, the boxed region is magnified in the bottom right inset. Magnified views of the individual channels are found in the insets under ZOOM. (B, C) MDCK cells were stained for endogenous TBC1D9B and either the (B) pIgR or (C) endogenous Sec15A. (D) Coefficient of colocalization for endogenous TBC1D9B and the indicated proteins. (E, F) MDCK cells were transfected with cDNA encoding GFP-Rab11a-WT (E) or GFP-Rab11a-QL (F) and after 72 h fixed and then stained for endogenous TBC1D9B. (G) Coefficient of colocalization for TBC1D9B and the indicated Rab11a construct. In (B, C, E, and F), the boxed region is magnified in the panels under the label ZOOM. Scale bar, 5 μm. In D and G, data were obtained from at least three independent experiments, and the means ± SEM are shown (n ≥ 150 cells). In G, a Student's t test was used to show that difference between these two sample groups was significantly different (*p < 0.05).
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Figure 7: Localization of TBC1D9B at sites of active Rab11a function. (A) Filter-grown MDCK cells were pulse labeled with 200 μg/ml IgA at the basolateral surface for 20 min at 18°C and chased for 20 min at 37°C in the presence of apical Cy5-labeled anti-IgA antibodies. The cells were fixed and then immunolabeled to detect endogenous TBC1D9B (green), endogenous Rab11a (red), and transcytosed IgA (blue). In the merged panel, the boxed region is magnified in the bottom right inset. Magnified views of the individual channels are found in the insets under ZOOM. (B, C) MDCK cells were stained for endogenous TBC1D9B and either the (B) pIgR or (C) endogenous Sec15A. (D) Coefficient of colocalization for endogenous TBC1D9B and the indicated proteins. (E, F) MDCK cells were transfected with cDNA encoding GFP-Rab11a-WT (E) or GFP-Rab11a-QL (F) and after 72 h fixed and then stained for endogenous TBC1D9B. (G) Coefficient of colocalization for TBC1D9B and the indicated Rab11a construct. In (B, C, E, and F), the boxed region is magnified in the panels under the label ZOOM. Scale bar, 5 μm. In D and G, data were obtained from at least three independent experiments, and the means ± SEM are shown (n ≥ 150 cells). In G, a Student's t test was used to show that difference between these two sample groups was significantly different (*p < 0.05).
Mentions: Next, we determined whether TBC1D9B was localized to vesicle populations where Rab11a was likely to be in its active, GTP-bound state. One such vesicle pool is the apical recycling endosome, which is accessed by the pIgR late in its transit from the basolateral-to-apical poles of the cell (Apodaca et al., 1994; Casanova et al., 1999; Wang et al., 2000a). As predicted, endogenous TBC1D9B colocalized with Rab11a and the basolaterally internalized pIgR ligand, IgA, in subapical vesicles (coefficient of colocalization of 0.59 ± 0.02; Figure 7, A and D). In addition, TBC1D9B colocalized with vesicles positive for the pIgR (coefficient of colocalization of 0.54 ± 0.07; Figure 7, B and D) or the Rab11a (and Rab8a) effector, Sec15A, one subunit of the octameric exocyst complex (coefficient of colocalization of 0.33 ± 0.04; Figure 7, C and D; Guo et al., 1999; Zhang et al., 2004; Wu et al., 2005; Novick et al., 2006; Oztan et al., 2007).

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus