Fig. 5.
Dynein/dynactin is nearly absent on the surface of retrolinkin-associated vesicles in BPAG1 null axons. (A and B) Vesicle fractionation assays. (A) Spinal cord and sciatic nerve tissue extracts from WT and BPAG1 null mice (KO) were centrifuged through an 8–35–40% sucrose gradient for 30 min at 150,000 × g. The interface pellets were collected and analyzed sequentially with antiretrolinkin (RTLN) and anti-Rab5B, anti-DIC, anti-LAMP-2, and anti-Rab7 (compare lanes 3 with 4). Endo, endosomal fraction; heavy, heavy membrane fraction. (B) After this prescreening, the endosomal fractions of WT and KO samples were further analyzed through a 5–10–15–20–40% sucrose gradient and immunoprobed with anti-p150Glued, anti-DIC, and Rab5B, confirming that the level of dynein/dynactin motor proteins in the KO samples were significantly decreased in endosomal fractions (compare lanes 5 and 6, and 7 and 8). F1, interface of 5–10% (light membrane fraction); F2, interface of 10–15% (early endosome rich fraction); F3, interface of 15–20% (early plus late endosomes). The 20–40% fraction was not taken. (C–I) Double immuno-EM analysis of sciatic nerve sections shows that the dynein motor protein associates with retrolinkin-labeled vesicles in WT (D–F) but fails to dock onto the same type of vesicles in BPAG1 null (G–I). Negative controls were performed with secondary antibodies alone in C. White arrows indicate colocalization of retrolinkin (15 nm) and DIC (5 nm). White boxes in H indicate retrolinkin labeling on fused and accumulated vesicles (high magnification in I), and the black arrow indicates DIC labeling (I) in BPAG1 null axons. [Scale bar: 100 nm (C–E), 50 nm (F), 65 nm (G and I), and 600 nm (H).]