Motor Neuron Disease-Associated Mutant Vesicle-Associated Membrane Protein-Associated Protein (VAP) B Recruits Wild-Type VAPs into Endoplasmic Reticulum-Derived Tubular Aggregates

The P56S mutation leads to aberrant aggregation of VAPA and VAPB. A–D, HeLa cells expressing HA-tagged VAPB-wt (A), VAPB-P56S (B, D), and VAPA-P56S (C) were fixed and processed for immunofluorescence using antibodies against HA (green) and the ER marker calreticulin (red). HeLa cells in D were incubated for 2 h at 20°C before fixation. Enlargement of boxed regions indicated in the merged pictures is shown at right. E, Lysates (L) of COS-1 cells transfected with HA-VAPB-wt and HA-VAPB-P56S were solubilized with Triton X-100, fractionated in supernatant (S), and pellet (P) under nonreducing (−DTT) or reducing (+DTT) conditions and analyzed by immunoblotting using anti-HA antibodies. Cells were either maintained at 37°C or incubated for 2 h at 20°C before lysis. F, Quantification of the number of VAPB-P56S clusters per cell, incubated at 15 or 20°C for 3 h or treated with 5 μg/μl BFA or 10 μm Nocodazole (Noco) for 30 min, as indicated. Error bars indicate SEM. **p < 0.005%. Scale bar, 10 μm.

Electron microscopy analysis of VAPB-P56S aggregates in HeLa cells. A1–C2, Transmission electron photomicrographs of anti-HA immunogold labeled ultrathin sections of HeLa cells transfected with HA-VAPB-P56S. Immunogold labeling is specifically associated with clusters of electron dense tubular profiles (A). Occasionally, labeled profiles are continuous with unlabeled profiles reminiscent of ER tubules (B) or with mitochondrial outer membranes (C). Arrows indicate unlabeled ER-tubules; asterisks indicate normal appearing cytoplasm in VAPB-P56S aggregates. m, Mitochondria; nu, nucleus. Scale bars: A1, B1, C1, 200 nm; A2, B2, C2, 100 nm.

VAPB-P56S clusters are immobile porous aggregates. A, Diagram of GFP-tagged VAPB constructs. B, Fluorescent recovery plots compare the rates of recovery of GFP-VAPB-wt (black triangle), GFP-VAPB-P56S (black box), as well as a nonbleached control region (open circles). Fluorescence intensity was normalized to intensity before bleaching. C–E, Representative confocal images of HeLa cells expressing GFP-VAPB-wt and GFP-VAPB-P56S. To visualize the mobility of the GFP-chimeras, small regions of interest of identical size (outlined square in C) were photobleached and monitored for recovery for 300 s (D). Half the area of a VAPB-P56S aggregate was bleached in E. F, Projection of confocal images of a HeLa cell expressing HA-VAPB-P56S and GFP, fixed and stained with anti-HA (red) antibody. Enlargement of the boxed region containing a single confocal stack (0.6 μm) is shown at the bottom. Scale bars, 10 μm.

VAPB-P56S recruits both wild-type VAPA and VAPB. A, B, HeLa cells transfected with HA-VAPB-P56S (A) or HA-VAPA-P56S (B) and stained with anti-HA (green) and anti-VAPA (red; A) or anti-VAPB (red; B) antibodies. Endog., Endogenous. C, D, HeLa cells double transfected with HA-VAPB-P56S and myc-VAPB-wt (C) or myc-VAPA-wt (D), fixed and stained with anti-HA (green) and anti-Myc antibodies (red) to visualize the expressing VAP protein. E, Lysates (L) of COS-1 cells transfected with HA-VAPB-wt and HA-VAPB-P56S were solubilized with Triton X-100, fractionated in supernatant (S) and pellet (P), and analyzed by immunoblotting using anti-VAPB (#1006-03) antibodies. F, COS-1 cells cotransfected with HA-VAPB-P56S and myc-VAPA-wt or myc-VAPB-wt were immunoprecipitated with control IgG or HA antibodies. The cells were incubated for 2 h at 20°C before lysis to solubilize VAPB-P56S. Each immunoprecipitation reaction is shown in three lanes: I, input to IP reaction; S, supernatant remaining after IP; P, precipitated pellet. The L, I, S, and P samples were immunoblotted for the indicated proteins. G, COS-1 cells cotransfected with HA-VAPB-P56S and GFP-VAPB-wt-N or GFP-VAPB-TMD were immunoprecipitated and immunoblotted as in F. H–K, HeLa cells single or double transfected with GFP-VAPB-TMD (H, J) or GFP-VAPB-wt-N (I, K) and HA-VAPB-P56S (J, K) and stained with anti-calreticulin to reveal the ER (H, I), or anti-HA (J, K) antibodies to visualize the expressing VAP protein. L–N, HeLa cells single or double transfected with GFP-VAPB-P56S-N and HA-VAPB-wt (M) or HA-VAPB-P56S (N) and stained with anti-calreticulin (L) or anti-HA (M, N) antibodies. Scale bar, 10 μm.

VAPB-P56S disturbs binding to FFAT motif-containing proteins. A–C, HeLa cells double transfected with GFP-FFAT plus HA-VAPB constructs and stained with anti-HA (red) antibodies to visualize the expressing VAP protein. D–F, HeLa cells double transfected with ORP3-GFP plus HA-VAPB constructs and stained with as in A–C. G, COS-1 cells cotransfected with GFP-FFAT and VAPB constructs were immunoprecipitated with GFP antibodies. Each IP reaction is shown in three lanes: I, input to IP reaction; S, supernatant remaining after IP; P, precipitated pellet. The I, S, and P samples were immunoblotted for the indicated proteins. H, COS-1 cells cotransfected with GFP-FFAT and HA-VAPB-wt were immunoprecipitated and immunoblotted as in G. I, Coomassie-stained gel of purified GST-VAPB proteins (top). The GST fusion proteins were loaded with HeLa-cell extracts transfected with GFP-FFAT and GFP-FFAT-SCR, precipitated, and immunoblotted (bottom two panels). Twenty percent of the input (I) is loaded on the gel. J, COS-1 cells cotransfected with ORP3-GFP and HA-VAPB constructs were immunoprecipitated with control IgG and GFP antibodies and immunoblotted as in G. Scale bar, 10 μm.

VAPB-P56S expression and VAP reduction causes cell death and Golgi fragmentation in primary neurons. A, B, Primary neurons expressing wild-type and mutant HA-tagged VAPA and VAPB were fixed and stained with HA-antibodies (green) and anti-PDI antibodies (red in B). Enlargement of the boxed region containing a single confocal stack (0.8 μm) is shown on the right. C, Primary neurons transfected with wild-type and mutant HA-tagged VAPB and shRNA against VAPA and VAPB (right) at DIV13 for 2 (left, middle) and 6 (right) d and stained with anti-HA (green; left, middle) or anti-β-galactosidase (green, right) and anti-PDI antibodies (red; insets). D, Representative images of primary neurons expressing wild-type (left) and mutant (middle) HA-tagged VAPB and shRNA against VAPA and VAPB (right), fixed and stained with HA-antibodies (green; left, middle) or β-galactosidase (right) and the cis-Golgi marker GM130 (red; insets). E, Quantification of neuronal survival by counting the number of wild-type VAPB-, mutant VAPB-, and β-galactosidase-expressing neurons per coverslip, 2 d (VAPB-WT and mutant) or 6 d (shRNA) after transfection from six and four independent experiments, respectively. F, Quantification of percentage of neurons with dispersed Golgi apparatus of control GFP, wild-type, or mutant VAPB- and shRNA-expressing cells 2 d (VAPB-WT and mutant) or 3 d (shRNA) after transfection. Scale bars: A, 10 μm; C, 100 μm ***p < 0.0005; **p < 0.001; *p < 0.10. Error bars indicate SEM.