HAP1 binds MTs independently of motors and is not required for autophagosome motility in the proximal axon. (A and B) Peroxisome recruitment assay showing peroxisome (PEX-GFP-DHFR) localization for 25 min following addition of dimerizer or DMSO. Left: Initial (0 min) and concluding (25 min) stills of peroxisomes. Right: Maximum time projection pseudo-colored by frame (scale above). Yellow line indicates cell outline. Scale bar, 10 µm. (C and D) Quantification and example micrographs from kinesin MT recruitment TIRF assay wherein cell extracts expressing constitutively active N-terminal kinesin construct K560 (positive control) or full-length kinesin-1 heavy chain (KHC) were added to Taxol-stabilized MT in a TIRF chamber with nonhydrolyzable ATP homologue AMPPNP. TMR-labeled kinesin intensity is measured across a 3-min video. n = 20 MT from four independent trials; one-way ANOVA with Tukey’s multiple comparisons test (KHC vs. K560, P = 0.0019; KHC vs. KHC + HAP1, P = 0.9992; KHC vs. KHC + HAP1 + Htt, P = 0.9833). Scale bar, 5 µm. (E and F) Blotting and quantification of cell extract MT pelleting assay for dynactin p150 (E) and dynein LIC1 (F) normalized to 5 µM MT only condition. n = 3–4 independent assays; one-way ANOVA with Tukey’s multiple comparisons test. p150 (ATP addition, P = 0.0463; KCl without ATP, P = 0.0025; KCl with ATP, P = 0.6624); DIC (ATP addition, P = 0.0264; KCl without ATP, P = 0.0025; KCl with ATP, P > 0.9999). (G) Immunoblot and quantification of KIF5 KD. Normalization factor (NF) determined using Revert Total Protein Stain). (H) Blotting and quantification of cell extract MT pelleting assay showing HAP1 binds MT independently of KIF5. Normalized to mock 5 µM MT with 1 mM Mg-ATP and no added salt; n = 3 independent assays; one-way ANOVA with Tukey’s multiple comparisons test (KD, P > 0.9999; KCl in mock, P = 0.0849; KCl in KD, P = 1662). (I) Example kymograph of LC3 in the proximal with Halo-HAP1WT. (J–M) Example kymographs and quantification showing no effect of HAP1 CC1 box or Glued motif mutants on LC3+ puncta motility in the proximal axon. n = 9–13 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde: AAVV, P = 0.1337; ID, P > 0.9999; EEAA, P = 0.5408; stationary/bidirectional [Stat/Bidir]: AAVV, P = 0.0900; ID, P > 0.9999; EEAA, P = 0.4316.) (N and O) Example kymograph and quantification showing no effect of HAP Spindly mutant in the proximal axon of cells transfected with HAP1 siRNA. n = 10–12 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde, P > 0.9999; Stat/Bidir, P > 0.9999). Bars throughout show mean ± SEM. *, P < 0.05; **, P < 0.01 compared to WT unless otherwise indicated.
Figure S3.

HAP1 binds MTs independently of motors and is not required for autophagosome motility in the proximal axon. (A and B) Peroxisome recruitment assay showing peroxisome (PEX-GFP-DHFR) localization for 25 min following addition of dimerizer or DMSO. Left: Initial (0 min) and concluding (25 min) stills of peroxisomes. Right: Maximum time projection pseudo-colored by frame (scale above). Yellow line indicates cell outline. Scale bar, 10 µm. (C and D) Quantification and example micrographs from kinesin MT recruitment TIRF assay wherein cell extracts expressing constitutively active N-terminal kinesin construct K560 (positive control) or full-length kinesin-1 heavy chain (KHC) were added to Taxol-stabilized MT in a TIRF chamber with nonhydrolyzable ATP homologue AMPPNP. TMR-labeled kinesin intensity is measured across a 3-min video. n = 20 MT from four independent trials; one-way ANOVA with Tukey’s multiple comparisons test (KHC vs. K560, P = 0.0019; KHC vs. KHC + HAP1, P = 0.9992; KHC vs. KHC + HAP1 + Htt, P = 0.9833). Scale bar, 5 µm. (E and F) Blotting and quantification of cell extract MT pelleting assay for dynactin p150 (E) and dynein LIC1 (F) normalized to 5 µM MT only condition. n = 3–4 independent assays; one-way ANOVA with Tukey’s multiple comparisons test. p150 (ATP addition, P = 0.0463; KCl without ATP, P = 0.0025; KCl with ATP, P = 0.6624); DIC (ATP addition, P = 0.0264; KCl without ATP, P = 0.0025; KCl with ATP, P > 0.9999). (G) Immunoblot and quantification of KIF5 KD. Normalization factor (NF) determined using Revert Total Protein Stain). (H) Blotting and quantification of cell extract MT pelleting assay showing HAP1 binds MT independently of KIF5. Normalized to mock 5 µM MT with 1 mM Mg-ATP and no added salt; n = 3 independent assays; one-way ANOVA with Tukey’s multiple comparisons test (KD, P > 0.9999; KCl in mock, P = 0.0849; KCl in KD, P = 1662). (I) Example kymograph of LC3 in the proximal with Halo-HAP1WT. (J–M) Example kymographs and quantification showing no effect of HAP1 CC1 box or Glued motif mutants on LC3+ puncta motility in the proximal axon. n = 9–13 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde: AAVV, P = 0.1337; ID, P > 0.9999; EEAA, P = 0.5408; stationary/bidirectional [Stat/Bidir]: AAVV, P = 0.0900; ID, P > 0.9999; EEAA, P = 0.4316.) (N and O) Example kymograph and quantification showing no effect of HAP Spindly mutant in the proximal axon of cells transfected with HAP1 siRNA. n = 10–12 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde, P > 0.9999; Stat/Bidir, P > 0.9999). Bars throughout show mean ± SEM. *, P < 0.05; **, P < 0.01 compared to WT unless otherwise indicated.

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