Figure 5. Panel A shows an illustration... | Rockefeller University Press

Figure 5.

Ca2+-dependent inhibition of kinesin-1–driven organelle motility. (A) Experimental design. Astrocytes co-express GCaMP6f, FRB-tagged organelles, and FKBP-tagged kinesin-1 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 < 0.0001. Error bars represent the mean ± SEM; n = 3 independent experiments. Refer to the image caption for details.

Panel A shows an illustration of the experimental design where astrocytes co-express GCaMP6f, FRB-tagged organelles, and FKBP-tagged Kinesin-1 for rapamycin-inducible motor recruitment. Panels B to E show kymographs and violin plots of organelle movement in single process regions of interest (ROIs) before and after the addition of rapamycin, along with corresponding calcium two positive amplitude traces. Panels F to I show kymographs and violin plots of organelle movement before and after the addition of DMSO, along with corresponding calcium two positive amplitude traces. Each kymograph displays the movement of organelles over time, with the x-axis representing time in seconds and the y-axis representing distance in micrometers. The calcium two positive amplitude traces show the changes in calcium two positive levels over time, with the x-axis representing time in seconds and the y-axis representing calcium two positive amplitude. The kymographs and traces illustrate the effects of rapamycin and DMSO on organelle motility and calcium two positive levels in astrocytes.

Ca ²⁺ -dependent inhibition of kinesin-1–driven organelle motility. (A) Experimental design. Astrocytes co-express GCaMP6f, FRB-tagged organelles, and FKBP-tagged kinesin-1 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 < 0.0001. Error bars represent the mean ± SEM; n = 3 independent experiments.