Figure 1: Release of LIS1 from a LIS1–dynein complex and activation of dynein motility by GTP-bound form of Rab6a. | Nature Communications

Figure 1: Release of LIS1 from a LIS1–dynein complex and activation of dynein motility by GTP-bound form of Rab6a.

From: Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement

Figure 1

(a) Summary of MT-gliding assay: percent of TMR-labelled MTs that displayed fluctuation (grey column) or gliding (black column). We used cytoplasmic dynein purified from porcine brain, which displayed approximately 0.5 μm s−1 average gliding velocity. Conditions are shown at the bottom of the graph. N: observed number of chambers. P-values were calculated using a Student’s t-test. (b) Immunoprecipitation assay using an anti-DIC antibody: protein combinations are shown at the top of the panels, and antibodies employed for western blotting (WB) are shown at the left side of the panels. (c) Quantitation of immunoprecipitation: precipitated DIC (left panel, N=3), LIS1 (middle panel, N=3) and Rab6a mutants (right panel, N=3), respectively, are shown. Each input for immunoblotting was expressed as 1.0. P-values were calculated using analysis of variance (ANOVA). (d) Immunoprecipitation assay using an anti-LIS1 antibody: applied protein combinations are shown at the top of the panels, and antibodies employed for WB are shown at the left side of the panels. (e) Quantitation of immunoprecipitation: precipitated LIS1 (left panel, N=3) and DIC (right panel, N=3) are shown. Each input for immunoblotting was expressed as 1.0. P-values were calculated using ANOVA. (f) MT pull-down assay using purified dynein, recombinant LIS1 and Rab6a(Q72L): protein combinations are shown at the bottom of the panels, and antibodies employed for WB are shown at the left side of the panels. (g) Quantitation of MT pull-down assay: co-precipitated dynein (left panel, N=6) and LIS1 (right panel, N=6) are shown. P-values were calculated using ANOVA. Note: Rab6a(Q72L) reduced dynein and LIS1, which were co-precipitated with MTs. (h) MT pull-down assay using dynein, LIS1 and Rab6a(T27N): protein combinations are shown at the bottom of the panels, and used antibodies for WB are shown at the left side of the panels. (i) Quantitation of MT pull-down assay: co-precipitated dynein (left panel, N=6) and LIS1 (right panel, N=6) are shown. P-values were calculated using ANOVA. All data expressed as mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001. N.S., not significant; ppt., precipitation; sup., supernatant.

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