JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin

Abstract

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).

Figure 1.

Binding of JRAB to Rab13. (A)Yeast transformants carrying the prey vector pACT2 encoding clone TTY124 and pGBDU vector encoding Rab1A WT, Rab5A WT, Rab13 WT, Rab13 T22N, Rab13 Q67L, or Rab13 N121I were spotted on synthetic complete medium lacking adenine to score for ADE2 reporter activity and incubated at 30°C for 3 d. (B) Structures of the full length and various fragments of JRAB. CH, calponin homology domain; LIM, LIM domain; CC, coiled-coil domain. (C) BHK cells were cotransfected with pCI-neo-Myc vector encoding JRAB-F, JRAB-N, or JRAB-C and pCI-neo-HA vector encoding Rab1A, Rab5A, or Rab13 as indicated. BHK cell extracts were immunoprecipitated (IP) with an anti-HA antibody and analyzed by Western blot (WB) with anti-Myc and anti-HA antibodies. The arrows indicate JRAB-F, JRAB-N, and JRAB-C. (D) BHK cells were cotransfected with pCI-neo-Myc-JRAB-F and either pCI-neo-HA-Rab13 T22N or pCI-neo-HA-Rab13 Q67L and were subjected to immunofluorescence microscopy using anti-Myc and anti-HA antibodies. Bar, 20 μm. The results shown in A, C, and D are representative of three independent experiments.

Figure 2.

Expression and localization of JRAB. (A) Homogenates of the indicated mouse tissues and cells (30 μg of protein each) were subjected to SDS-PAGE, followed by Western blot with an anti-JRAB antibody. The PVDF membranes stained with Coomassie brilliant blue (CBB) are presented in the bottom panels. (B) Subcellular fractionation of MTD-1A cells was performed and the indicated fractions were subjected to SDS-PAGE, followed by Western blot with an anti-JRAB antibody. (C) MTD-1A cells were double immunostained with anti-JRAB and anti-ZO-1 antibodies and observed under a confocal microscope. Vertical sectional images were generated and shown in the bottom panels. In the merged images, JRAB was green and ZO-1 was red. Bars, 10 μm. ap, level of apical membranes. The results shown are representative of three independent experiments.

Figure 3.

Involvement of JRAB in the endocytic recycling of occludin in MTD-1A cells. (A) MTD-1A cells infected with Ad-EGFP (GFP), Ad-Myc-JRAB-F (JRAB-F), Ad-Myc-JRAB-N (JRAB-N), Ad-Myc-JRAB-C (JRAB-C), or Ad-EGFP-Rab13 Q67L (Rab13 Q67L) were cell surface biotinylated and incubated at 37°C for 15 min to allow for endocytosis of biotinylated occludin or TfR on the cell surface. After stripping the remaining biotin from the cell surface, cells were again incubated at 37°C for 15 min to allow recycling of biotinylated occludin or TfR back to the cell surface. After a second stripping of cell surface biotin, biotinylated occludin or TfR was isolated with avidin beads and quantitated by Western blot with anti-occludin or anti-TfR antibody. Recycled proteins represent the percentage of endocytosed proteins and expressed as the mean and SEM of three independent experiments. (B) MTD-1A cells infected with Ad-EGFP (GFP) or Ad-Myc-JRAB-N (JRAB-N) were cell surface biotinylated and incubated at 37°C for 15 min to allow for endocytosis of biotinylated occludin or TfR on the cell surface. After stripping the remaining biotin from the cell surface, biotinylated occludin or TfR was isolated and quantitated. Endocytosed proteins represent the percentage of endocytosed proteins and expressed as the mean and SEM of three independent experiments.

Figure 4.

Involvement of JRAB in the recruitment of occludin to the PM during Ca²⁺ switch in MTD-1A cells. MTD-1A cells infected with Ad-EGFP (GFP), Ad-Myc-JRAB-N (JRAB-N), or Ad-EGFP-Rab13 Q67L (Rab13 Q67L) were subjected to the Ca²⁺ switch assay, and were then double immunostained with anti-occludin and anti-GFP antibodies at 0, 4, 6, and 24 h after Ca²⁺ restoration. Bar, 20 μm. The results shown are representative of three independent experiments.

Figure 5.

Involvement of JRAB in the development of TER during Ca²⁺ switch in MTD-1A cells. MTD-1A cells expressing GFP (GFP), Myc-tagged JRAB-N (JRAB-N), or GFP-tagged Rab13 Q67L (Rab13 Q67L) were subjected to Ca²⁺ switch assay. TER was measured at 0, 12, 24, and 48 h after Ca²⁺ restoration. The results shown are the mean and SEM of three independent experiments performed in duplicate.

Figure 6.

Involvement of JRAB in the formation of paracellular diffusion barrier during Ca²⁺ switch in MTD-1A cells. MTD-1A cells expressing GFP (GFP), Myc-tagged JRAB-N (JRAB-N), or GFP-tagged Rab13 Q67L (Rab13 Q67L) were subjected to Ca²⁺ switch assay. Paracellular fluxes of 4- and 40-kDa FITC-dextran were measured at 12 h after Ca²⁺ restoration. The amount of tracer diffusion is normalized to that of GFP-expressing MTD-1A cells. The results shown are the mean and SEM of three independent experiments performed in duplicate.

Figure 7.

Requirement of JRAB localization to TJs. (A) MTD-1A cells were transfected with pCI-neo-HA-Rab13 WT, immunoprecipitated (IP) with an anti-HA antibody, and analyzed by Western blot (WB) with anti-JRAB and anti-HA antibodies. The arrows indicate endogenous JRAB and HA-Rab13 WT. The double stars indicate the Ig light chain. (B) MTD-1A cells were cotransfected with pCI-neo-Myc-JRAB-F and pCI-neo-HA-Rab13 WT, pCI-neo-HA-Rab13 T22N, pCI-neo-HA-Rab13 Q67L, or pCI-neo-HA-Rab13 N121I and were subjected to immunofluorescence microscopy using anti-Myc and anti-HA antibodies. In the merged images, JRAB was green and Rab13 was red. Bar, 20 μm. (C) MTD-1A cells were infected with Ad-Myc-JRAB-F (JRAB-F), Ad-Myc-JRAB-N (JRAB-N), or Ad-Myc-JRAB-C (JRAB-C) and subjected to immunofluorescence microscopy using an anti-Myc antibody. Bar, 20 μm. (D) MTD-1A cells were incubated for 2 h in the absence (control) or presence of 10 μM cytochalasin D (CytoD) and double labeled with anti-JRAB antibody and rhodamine-phalloidin. The arrow indicates a circumferential actin belt. In the merged images, JRAB was green and F-actin was red. Bar, 20 μm. (E) MTD-1A cells were incubated in Ca²⁺-chelated medium for 60 min and double-labeled with anti-JRAB antibody and rhodamine-phalloidin. The arrow indicates radiating actin cables. In the merged images, JRAB was green and F-actin was red. Bar, 20 μm. (F) NIH3T3 cells were double labeled with anti-JRAB antibody and rhodamine-phalloidin. The arrow indicates stress fibers. In the merged images, JRAB was green and F-actin was red. Bar, 10 μm. The results shown in A-F are representative of three independent experiments.