Dynein-dynactin localization and retrograde movement are intact in actr10 mutants.
(A,B) Dynein heavy chain (DHC) immunolabeling demonstrates normal DHC localization in NM3 axon terminals of actr10 mutants at 4 dpf. (C,D) p150 is normally localized in NM3 axon terminals of actr10 mutants at 4 dpf. (E,F) Analyses of mean fluorescence intensity in axon terminals (background subtracted) showed comparable fluorescence intensity between mutant and wildtype siblings (ANOVA; mean ± SEM shown). (G,H) Stills from dynein time-lapse imaging sessions (Videos 3 and 4) in wildtype and actr10 mutant pLL axons at 4 dpf. Arrowheads indicate retrograde dynein movement. (I,J) Kymograph analyses of dynein transport in wildtype (I) and actr10 mutants (J). (K–M) Retrograde dynein transport parameters, including the proportion of dynein-labeled vesicles (K), distance moved by vesicles (L) and velocity of movement (M) are unaffected in actr10 mutants (ANOVA; mean ± SEM shown). A reduction of anterograde dynein-positive puncta distance and velocity was noted (ANOVA; mean ± SEM shown). (N) Dynein intermediated chain 2b fused to GFP (i2b-GFP) interacts with the core dynein complex. Immunoprecipitation of i2b-GFP from whole embryo extracts followed by DHC western (top). Whole embryo lysate controls for i2b-GFP (middle; top band not present in wildtype) and DHC (bottom). Bead only (no 1°) immunoprecipitation controls shown. (O) The core dynein complex labeled by i2b-GFP can immunoprecipitate p150 from whole larval lysates derived from wildtype and actr10 mutant larvae at 4 dpf (top). Whole larval lysate control for i2b-GFP (middle) and p150 (bottom). Bead only (no 1°) and un-injected wildtype immunoprecipitation controls shown. Scale bars = 10 µm. Number of larvae assayed is indicated on graphs.