Characterization of BPAG1n4. (A) Schematic of the domain structures of BPAG1 isoforms. I, first exon of each isoform; BPAG1e, the epithelial isoform; ABD, actin-binding domain; IF-BD, intermediate filament-binding domain; M1, microtubule-binding domain. Amino acid residue numbers denote functional domain boundaries in BPAG1n4. Individual domains and regions used in this work are illustrated below. Anti-BPAG1n recognizes all BPAG1 isoforms. (B) Northern blot analysis of mouse dorsal root ganglion (DRG) RNA. (probes) A 1.6-kb cDNA head domain fragment (top; 4-d exposure); GAPDH as internal control (bottom; 1-h exposure). (C) Protein expression of BPAG1n4 in mouse brain. Blot was probed with anti-BPiso4. COS-7 (lane 3) and NIH 3T3 cells (lane 4) were transfected with an expression construct for n4-92 (A, bottom) encompassing the epitope for anti-BPiso4. (untrans.) Untransfected COS-7 (lane 5) and NIH cells (lane 6). (D–E) ImmunoEM reveals subcellular localization of BPAG1n4 in dorsal roots. (D) BPAG1n4 labeling with anti-BPiso4 was visualized with 12-nm gold particles conjugated with secondary antibody. White arrows indicate BPAG1n4 on vesicle-like structures in association with microtubules. (E) Negative control, secondary antibodies alone. Bar: (D) 300 nm; (E) 400 nm.

Colocalization of BPAG1n4 with dynactin/dynein and up-regulation of p150^Glued in BPAG1 null mouse. (A–D) Double immunofluorescence staining of cultured DRG neurons shows colocalization of BPAG1n4 with dynactin/dynein. Cells were stained with anti-BPiso4/anti-p150^Glued (BD Biosciences) in A–C and anti-BPiso4 /anti-DIC (Chemicon) in D. AlexaFluor-conjugated secondary antibodies (Molecular Probes) were used for detection. Insets show colocalizations at higher magnification. (E) Double ImmunoEM labeling of mouse sciatic nerve sections with anti-BPiso4 (18 nm) and anti-DIC (6 nm). Arrows indicate colocalization of BPAG1n4 and dynein. (F) Negative control with secondary antibodies only. (G) RT-PCR analysis of DRG mRNAs. Multiplex gene-specific primers (lanes 1 and 2) were used on cDNAs from WT and null DRG. Lanes 3–6 indicate each individually amplified primer pair product from WT: p150^Glued (410 bp), microtubule associated protein 1B light chain (MAP1B-LC; 850 bp), gigaxonin (Gig, 780 bp), and actin (560 bp). Arrowhead indicates up-regulation of p150^Glued in BPAG1 null (lane 2) versus WT (lane 1). (H) Protein analysis on mouse spinal cord and sciatic nerve lysates using antibodies to p150^Glued, DIC, and p50 (BD Biosciences). Antitubulin served as loading control. Bars: (A–D) 10 μm; (E and F) 400 nm.

Disruption of retrograde transport is BPAG1n4 isoform specific. Mouse DRG neurons were transfected with pEGFP-ERM-N1 or pEGFP-N1. Retrograde transport of transferrin conjugated with Texas red (Tf-TR) was examined as described in Materials and methods. Cells expressing ERM-GFP (A) or GFP (B) were first incubated with Tf-TR for 2 h. After removal of the Tf-TR, the cells were observed and pictured every 2 h. Shown are the cells after 2 h (E–H), 6 h (I–L), and 12 h (M–P). Untransfected DRG neurons of WT (C, G, K, and O) and BPAG1 null mice (D, H, L, and P) served as controls. The ERM-GFP–expressing neurons mimicked BPAG1 null neurons in their failure to transport Tf-TR retrogradely into the cell bodies. Bar, 23 μm.

Blot overlay and co-IP assays show BPAG1n4–dynactin interaction. (A) Blot overlay assays with ERM domain. 85-kD epitope-tagged ERM (HA-ERM), 78-kD gigaxonin (HA-Gig), or 90-kD BPAG1n4 COOH terminus (FLAG-n4-C-long) from transfected CHO cells were immobilized on membranes. Membranes were incubated with (+) or without (−) total lysates of mouse spinal cord and sciatic nerve and probed with anti-HA (HA.11; CRP, Inc.), anti-FLAG (Sigma-Aldrich), and anti-p150^Glued, respectively. In addition to its endogenous band (arrowhead), p150^Glued was also detected at the position of HA-ERM (lane 4, arrow) as a result of its binding to the ERM domain of BPAG1n4. (B) Blot overlay assays with BPAG1n4. BPAG1n4 was immobilized on membrane and overlaid with (+) or without (−) total lysates of spinal cord and sciatic nerve. Blots were probed with anti-p150^Glued and antigigaxonin (Gig, control). (lys) total brain lysates. p150^Glued (lane 3), but not gigaxonin (lane 6), binds to full-length BPAG1n4 (arrows). (C) Co-IP of ERM domain and p150^Glued in COS-7 cells. (WB) Western blotting; (IP) immunoprecipitation. Cell lysates of COS-7 transfected with pHA-ERM (lanes 2 and 3), or pFLAG-p150^Glued (lane 1), or both (lane 4) were incubated with anti-FLAG M2 affinity gel. Protein samples were analyzed by immunoblotting with anti-HA antibody. (D–E) co-IP of BPAG1n4 and the dynactin–dynein complex from mouse nerve tissue extracts. A rabbit anti–p150^Glued (Santa Cruz Biotechnology, Inc.) antibody, but not the sham antibody, was able to coimmunoprecipitate BPAG1n4 (D, lane 3). Conversely, anti-BPiso4 but not the sham antibody was able to coimmunoprecipitate the dynactin complex (E, lane 2). Antibodies against BPAG1n4, p150^Glued, p50, DIC, and control protein (sham) were used for immunoblotting. (F) Neither anti-p150^Glued nor anti-BPiso4 could coimmunoprecipitate other BPAG1 neuronal isoforms (BPAG1n1–3). Immunoblot was probed with rabbit antibody against the common rod domain shared by BPAG1n1–3 (anti-BProd).

Retrograde axonal transport is disrupted in BPAG1 null mice. (A and B) EM of longitudinal sections of spinal cord dorsal column reveals accumulation of vesicles and other organelles in BPAG1 null (A), but not in WT (B). (C–E) Proteins failed to accumulate on the distal side of a double ligature (schematic shown in C) of BPAG1 null sciatic nerves. Shown are confocal images of nerve sections stained with anti-p75^NTR (D), and immunoblots of p75^NTR from ∼3-mm sections proximal or distal to double ligatures (E). Anti–α-tubulin served as loading control. Bar: (A and B) 500 nm; (D) 140 μm.