Figure 4. Panel A shows a schematic of... | Rockefeller University Press

Figure 4.

Ca2+-dependent inhibition of dynein/dynactin-driven organelle motility. (A) Experimental design. Astrocytes co-express GCaMP6f, FRB-tagged organelles, and FKBP-tagged dynein/dynactin for rapamycin-inducible motor recruitment. (B–E) Rapamycin (100 nM) acutely increases organelle motility. Kymographs from single process ROIs show accelerated movement after rapamycin addition (B’–E’), together with corresponding Ca2+ amplitude traces. Subsequent global Ca2+ waves (10-s illumination) halted motility (B’’–E’’). N/A: Ca2+ intensity was not measured. Scale bars: 10 μm (kymographs), 1 μm (zoom in). (F–I) DMSO as controls. Kymographs show organelle movement (F’–I’) and Ca2+ amplitude traces before and after DMSO addition, followed by Global illumination. DMSO resulted in no change in motility, while global Ca2+ waves still trigger organelle arrest (F’’–I’’). N/A: Ca2+ intensity was not measured. Scale bars: 10 μm (kymographs), 1 μm (zoom in). (B’’’–I’’’) Quantification of mean organelle velocity (μm/s) in the same process segments at baseline, after rapamycin or DMSO treatment, and during global Ca2+ waves, analyzed with TrackMate. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test, ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Error bars represent the mean ± SEM; n = 3 independent experiments. Refer to the image caption for details.

Panel A shows a schematic of the organelle, FKBP–FRB interaction via rapamycin versus DMSO control, affecting BicaD2, kymograph output, organelle movement, and calcium amplitude. Panels B to E show kymographs, violin plots, and calcium amplitude traces for rapamycin-treated cells, with kymographs displaying organelle movement over time and amplitude traces showing changes in calcium levels before and after treatment. Panels F to I show similar data for DMSO-treated control cells, including kymographs, violin plots, and calcium amplitude traces. Each kymograph is labeled with time in seconds and distance in micrometers, while the calcium amplitude traces display calcium signal intensity over time. The violin plots quantify mean organelle velocity analyzed with TrackMate, with statistical significance indicated by asterisks. The plots show that rapamycin increases organelle motility, which is halted by global calcium waves, whereas DMSO does not alter motility, although global calcium waves still trigger organelle arrest.

Ca ²⁺ -dependent inhibition of dynein/dynactin-driven organelle motility. (A) Experimental design. Astrocytes co-express GCaMP6f, FRB-tagged organelles, and FKBP-tagged dynein/dynactin for rapamycin-inducible motor recruitment. (B–E) Rapamycin (100 nM) acutely increases organelle motility. Kymographs from single process ROIs show accelerated movement after rapamycin addition (B’–E’), together with corresponding Ca²⁺ amplitude traces. Subsequent global Ca²⁺ waves (10-s illumination) halted motility (B’’–E’’). N/A: Ca²⁺ intensity was not measured. Scale bars: 10 μm (kymographs), 1 μm (zoom in). (F–I) DMSO as controls. Kymographs show organelle movement (F’–I’) and Ca²⁺ amplitude traces before and after DMSO addition, followed by Global illumination. DMSO resulted in no change in motility, while global Ca²⁺ waves still trigger organelle arrest (F’’–I’’). N/A: Ca²⁺ intensity was not measured. Scale bars: 10 μm (kymographs), 1 μm (zoom in). (B’’’–I’’’) Quantification of mean organelle velocity (μm/s) in the same process segments at baseline, after rapamycin or DMSO treatment, and during global Ca²⁺ waves, analyzed with TrackMate. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test, ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Error bars represent the mean ± SEM; n = 3 independent experiments.