Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex.

Loss of TBC1D5 expression enhances endosomal levels of the retromer CSC. (A) HeLa cells were transiently transfected with empty GFP vector, GFP-TBC1D5 wild type (WT) or GFP-TBC1D5 R169A/Q204A (RQ) mutant. After fixation, the cells were stained with antibodies against VPS35 and VPS26. Transfected cells are marked with an asterisk. Overexpression of the wild-type TBC1D5 can displace the retromer CSC from membranes. (B) HeLa cells were treated with siRNA to silence TBC1D5 expression. The knockdown cells were mixed with control cells and seeded onto coverslips. After fixation, cells were labelled with anti-TBC1D5 and antibodies against either VPS35 or VPS26. Loss of TBC1D5 expression (in cells marked with an asterisk) results in brighter staining of the retromer CSC proteins. (C,D) HeLa cells treated with siRNA to silence TBC1D5 expression were labelled with antibodies against VPS26, TBC1D5, CIMPR or TGN46 and then imaged using an automated microscope. Loss of TBC1D5 results in ∼40% increase in VPS26 fluorescence intensity but does not markedly increase the number of VPS26-positive spots (D). No spots were counted for TGN46 as the morphology of the TGN is not punctate but ribbon-like. P-values for TBC1D5 knockdown versus control: VPS26, 1.2×10−4; CIMPR, 0.0095; TGN46, 0.0037 for total intensity values. The P-values for spot numbers are: 0.08 for VPS26 and 0.23 for CIMPR. Scale bars: 20 µm.

Loss of TBC1D5 function leads to increased levels of active Rab7a. (A) HeLa cells stably expressing GFP-tagged Rab7a-GTP were treated with siRNA to silence TBC1D5 expression. Following fixation, the cells were labelled with anti-Rab7a-GTP antibodies. Knockdown of TBC1D5 leads to increased staining of the anti-Rab7a antibody. Scale bar: 20 µm. (B) Cells stably expressing various GFP-tagged proteins were treated as in A and then imaged using an automated microscope. Only cells expressing GFP-Rab7a or GFP-Rab7a Q67L registered significant fluorescence and the knockdown of TBC1D5 results in a pronounced increase in the levels of active (GTP-bound) Rab7a. Values are mean±s.e.m. of 250 cells measured for each cell line. The P-values for control and TBC1D5 knockdown for cells expressing GFP-Rab7a are shown on the graph and demonstrate that the increase in fluorescence is statistically significant. (C) Cells expressing various GFP-tagged proteins were treated with siRNA to abolish either Rab7a or TBC1D5 expression. After fixation, the cells were labelled with antibodies against VPS26 and imaged using an automated microscope. Loss of Rab7a expression causes VPS26 (and the retromer CSC) to dissociate from endosomes, massively reducing the fluorescence intensity except where the knockdown of Rab7a is rescued by GFP-tagged Rab7a or Rab7a Q67L. Loss of TBC1D5 expression enhances VPS26 fluorescence even in cells where VPS26 fluorescence is lower due to the expression of a GDP-locked Rab7a T22N mutant. P-values are shown on the graph. (D) The number of VPS26 spots is not significantly altered after loss of TBC1D5 expression. P-values for all control versus TBC1D5-knockdown measurements were >0.1.

TBC1D5 knockdown enhances the associations of the retromer CSC. (A) Cells expressing either GFP-Rab5 or GFP-Rab7a were treated with siRNA to silence TBC1D5 expression. After lysis under native conditions, the GFP-tagged proteins were recovered by immunoprecipitation (IP) using anti-GFP. The immunoprecipitated proteins were analysed by western blotting. Retromer CSC proteins were detected in the GFP-Rab7a IP but not GFP-Rab5 IP. The knockdown of TBC1D5 increased the levels of retromer CSC proteins associated with GFP-Rab7a but no changes in protein levels were observed after TBC1D5 knockdown when lysates were analysed. IP of VPS26 confirmed that TBC1D5 expression was abolished and that loss of TBC1D5 does not alter the interaction between VPS26 and VPS35. (B) Following a protocol for SILAC labelling, three HeLa cell lines were labelled over several passages with heavy or light amino acids before being subjected to TBC1D5 knockdown (in the light amino acid-labelled cells) and treated with the DSP crosslinking reagent. After lysis, VPS26 was recovered by IP and the resulting IPs analysed by mass spectrometry. Levels of the proteins in the IPs are shown as a ratio of heavy:light normalised to VPS26. The retromer CSC proteins all give values close to 1, indicating that TBC1D5 knockdown does not affect retromer CSC assembly. The TBC1D5 protein has a ratio ∼3-3.5 consistent with a knockdown of the protein. Other proteins detected generally gave ratios <1, showing that more peptides labelled with light amino acids were detected, indicating an increased level of the protein after TBC1D5 knockdown. (C) Cells expressing either GFP-VPS35 WT or GFP-VPS35 D620N (DN) were treated as in B and the lysates were treated with anti-GFP antisera. The knockdown of TBC1D5 can enhance the interaction of the VPS35 D620N mutant with Fam21. (D) Lysates from cells in C analysed by western blotting to confirm TBC1D5 knockdown.

Silencing TBC1D5 expression can rescue the impaired Fam21 interaction of the VPS35 D620N mutant. (A,B) Cells expressing either wild-type GFP-VPS35 (WT) (A) or GFP-VPS35 D620N (DN) (B) were treated with siRNA to abolish TBC1D5 expression. After fixation, the cells were labelled with antibodies against Fam21 and the CIMPR. There is a marked increase in the fluorescence staining of Fam21 in the GFP-VPS35 D620N cells after TBC1D5 knockdown. Scale bar: 20 µm. (C) Cells from A and B were imaged using an automated microscope to measure the fluorescence intensity. Knockdown of TBC1D5 can rescue the fluorescence intensity of Fam21, consistent with an increase in the association of Fam21 (and the WASH complex) with the retromer CSC. P-values for TBC1D5 KD versus control are: VPS35 WT: VPS26, 2.7×10−6; CIMPR, 1.4×10−6; Fam21, 0.0022; VPS35 D620N: VPS26, 3.0×10−8; CIMPR, 9.8×10−6; Fam21, 8.5×10−9.

Inhibition of TBC1D5 function can partially rescue the trafficking defects caused by the VPS35 D620N mutation. (A) HeLa cells expressing GFP-VPS35 D620N were treated with siRNA to silence TBC1D5 expression. After fixation, the cells were labelled with antibodies against Glut1 and the CIMPR. The localisation of Glut1 appears to be shifted away from perinuclear structures after TBC1D5 knockdown. Scale bar: 20 µm. (B) HeLa cells expressing either GFP-VPS35 wild type (WT) or the GFP-VPS35 D620N mutant were treated with siRNA to silence TBC1D5 expression and then labelled with antibodies against Glut and Snx1. The cells were imaged using an automated microscope and the overlap of the Glut1 and Snx1 antibodies measured. There is more overlap of Glut1 with Snx1 in cells expressing VPS35 D620N. The overlap is reduced upon TBC1D5 knockdown but not to levels seen in cells expressing wild-type VPS35. (C) There is no appreciable change in the Snx1 fluorescence following TBC1D5 knockdown. (D) Cells treated as in B were labelled with anti-Glut1 and anti-GM130. (E) There is no appreciable change in GM130 fluorescence after TBC1D5 knockdown. For B-E, values are mean±s.d. and P-values are shown on the graphs.

Knockdown of TBC1D5 can enhance retromer function. (A) Three different cell lines were treated with siRNA to knockdown TBC1D5 expression. Following fixation, cells were labelled with antibodies against CIMPR and TGN46 and then imaged using an automated microscope. The fraction of CIMPR present in a TGN46 mask is shown graphically. For each of the cell lines, knockdown on TBC1D5 enhances the colocalisation of CIMPR with TGN46 but only the GFP-VPS35 cells demonstrate statistical significance. Values are mean±s.d. and P-values for knockdown versus control are shown for each cell line. (B) TBC1D5 expression was silenced in cells expressing GFP-VPS35 wild type or the D620N mutant. Following fixation, the cells were labelled with antibodies against Atg9A and TGN46 and then imaged using an automated microscope. The Pearson correlation coefficient for Atg9A-TGN46 mask is shown graphically. For each of the cell lines, knockdown of TBC1D5 enhances the colocalisation of Atg9A with TGN46 but only the GFP-VPS35 wild-type cells demonstrate statistical significance. Values are mean±s.d. and P-values for knockdown versus control are shown for each cell line. (C) HEK293 cells stably expressing APPswedish were treated with siRNA to silence VPS35 or TBC1D5. Cell culture medium was collected and analysed for the Aβ peptide by western blotting. Knockdown of VPS35 increases APP processing to Aβ but loss of TBC1D5 expression has the opposite effect. The data shown are from two independent experiments that were highly reproducible. Values are mean±s.d. For both the VPS35- and TBC1D5-knockdown conditions, P<0.01 using Student's t-test compared with control. (D) Representative blots of media (for Aβ and sAPPβ) and lysates (for APP, VPS35, TBC1D5 and the loading controls, GAPDH and tubulin) from C showing the reduction in Aβ detected when TBC1D5 is silenced. There is also a reduction in sAPPβ.