TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells

Abstract

Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity in vitro. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.

Fig 1. Identification of TBC proteins that specifically localize on recycling endosomes in MEFs.

(A) Typical images of four TBC proteins that clearly colocalized with TfR (a recycling endosome marker). MEFs transiently expressing EGFP-TBC proteins were immunostained with anti-TfR antibody (1/250 dilution), and the stained cells were examined with a confocal fluorescence microscope. The insets show magnified views of the boxed areas. The line scan profiles (broken arrows in the far right column) were acquired by using ImageJ software. (B) A typical image of TBC protein showing no colocalization with TfR. Scale bars, 40 μm. The images of all other TBC proteins are summarized in S1 Fig.

Fig 2. Rab11 recruits TBC1D12 to Rab11-positive recycling endosomes.

(A) Overexpression of Rab11 altered TBC1D12 localization from around the nucleus to the cell protrusions (arrowheads). MEFs transiently expressing both EGFP-TBC1D12 and mStr-Rab11A (or mStr alone as a control) were examined with a confocal fluorescence microscope. Scale bars, 10 μm. (B) Knockdown of Rab11A/B caused dispersion of TBC1D12 into the cytosol. MEFs stably expressing T7-TBC1D12 were transfected with control siRNA (siControl) or Rab11A/B siRNAs (siRab11A/B) and immunostained with anti-TBC1D12 antibody (1/300 dilution) and anti-Rab11 antibody (1/200 dilution), and the stained cells were examined with a confocal fluorescence microscope. Scale bars, 20 μm. (C) Knockdown of Rab11A/B in MEFs as revealed by immunoblotting. The band intensity of Rab11 in siRab11A/B-treated cells was 6.7% of its band intensity in the control cells. Total cell lysates of siControl-treated cells (lane 1) and siRab11A/B-treated cells (lane 2) were analyzed by 8% SDS-PAGE and immunoblotting with anti-GFP antibody (top panel; 1/500 dilution), anti-Rab11 antibody (middle panel; 1/500 dilution), and anti-β-actin antibody (bottom panel; 1/15,000 dilution). The positions of the molecular mass markers (in kDa) are shown on the left.

Fig 3. TBC1D12 interacts with Rab11 through its middle region.

(A) TBC1D12 interacts with a constitutively active form that mimics the GTP-bound form of Rab11. Total lysates of COS-7 cells expressing T7-TBC1D12 were incubated with glutathione-Sepharose beads coupled with GST alone, GST-Rab11A-Q70L (indicated by QL), or GST-Rab11A-S25N (indicated by SN). A 2% volume of the reaction mixture (input) and proteins bound to the glutathione-Sepharose beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top and middle panels), and the GST-fusion proteins were stained with amido black (bottom panel). The positions of the molecular mass markers (in kDa) are shown on the left. (B) Schematic representation of three deletion mutants of TBC1D12 (TBC1D12-N, -M, and -TBC). TBC domains and coiled-coil (CC) domains are shown as blue boxes and green boxes, respectively. (C) Total lysates of COS-7 cells expressing T7-TBC1D12 (indicated by FL, full-length), T7-TBC1D12-N, T7-TBC1D12-M, or T7-TBC1D12-TBC were incubated with glutathione-Sepharose beads coupled with GST alone (indicated by G) or GST-Rab11A-Q70L (indicated by 11). A 2% volume of the reaction mixture (input) and proteins bound to the beads were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-T7 tag antibody (top three panels). The third panel corresponds to the longer exposure of the second panel. The GST-fusion proteins were stained with amido black (bottom panel). The asterisk indicates non-specific bands, and the positions of the molecular mass markers (in kDa) are shown on the left.

Fig 4. Overexpression of TBC1D12 in PC12 cells promotes neurite outgrowth independently of its GAP activity.

(A) Overexpression of either TBC1D12 wild-type (WT) or its GAP-activity-deficient mutant (RK) promoted neurite outgrowth of PC12 cells. Typical images of PC12 cells expressing EGFP alone (control), EGFP-TBC1D12-WT, or EGFP-TBC1D12-RK. After NGF stimulation for 36 h the cells were fixed, immunostained with anti-GFP antibody (1/500 dilution), and examined with a fluorescence microscope. Scale bars, 30 μm. (B) The total neurite length of each cell in (A) after NGF stimulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. No significant difference was observed between the WT and RK mutant. (C) TBC1D12 promotes neurite outgrowth in a Rab11-dependent manner. Typical images of PC12 cells expressing EGFP alone (control) or EGFP-TBC1D12-WT together with siControl or siRab11A/B. After NGF stimulation for 36 h the cells were fixed, immunostained with anti-GFP antibody (1/500 dilution), and examined with a fluorescence microscope. Scale bars, 30 μm. (D) The total neurite length of each cell in (C) after NGF stimulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001; NS, not significant; and a.u., arbitrary unit. (E) Rab11-S25N (SN), but not Rab11-Q70L (QL), inhibits neurite outgrowth of both control cells and TBC1D12-expressing cells. Typical images are shown in S7 Fig. The total neurite length of each cell after NGF stimulation for 36 h was measured as described in (D). Error bars indicate the SEMs of the data from ≥79 cells. ***, p <0.001; **, p <0.01; NS, not significant. Although both positive and negative effects of Rab11-Q70L (or a positive effect of S25N) on neurite/axon outgrowth have been reported previously [10, 39], under our experimental conditions expression of Rab11-Q70L and Rab11-S25N in PC12 cells promoted and inhibited, respectively, NGF-stimulated neurite outgrowth.

Fig 5. Knockdown of TBC1D12 in PC12 cells promotes neurite outgrowth.

(A) Knockdown of TBC1D12 in PC12 cells promoted neurite outgrowth. Typical images of PC12 cells expressing EGFP together with either siControl, siTBC1D12 #1, or siTBC1D12 #2. After NGF stimulation for 36 h the cells were fixed and examined with a confocal fluorescence microscope. Scale bars, 30 μm. The knockdown efficiency of siTBC1D12 #1/2 is shown in S5C Fig. (B) The total neurite length of each cell in (A) after NGF simulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. *, p <0.05; and a.u., arbitrary unit. (C) Promotion of neurite outgrowth of TBC1D12-KD cells occurred in a Rab11-dependent manner. Typical images of PC12 cells expressing EGFP together with either siControl, siTBC1D12 #1 + siControl, siTBC1D12 #1 + siRab11A/B, or siRab11A/B. After NGF stimulation for 36 h the cells were fixed and then examined with a fluorescence microscope. Scale bars, 30 μm. (D) The total neurite length of each cell in (C) after NGF stimulation for 36 h was measured with MetaMorph software (n >100). The total neurite length of each sample was normalized to that of the control cells. Error bars indicate the SEMs of the data from three independent experiments. ***, p <0.001; NS, not significant; and a.u., arbitrary unit.