Panel A shows a schematic diagram of a neuron with a region of interest (ROI) used for calcium ion amplitude extraction, kymograph generation, merged kymograph, and organelle movement, with resting and global calcium ion signals indicated by different colors. Panels B to G each include three components: a line graph of calcium ion amplitude over time, a kymograph showing organelle movement, and corresponding time-lapse images. The line graphs have time in seconds on the x-axis and calcium ion amplitude on the y-axis, illustrating fluctuations in intracellular calcium levels. The kymographs represent organelle movement over time, where magenta arrowheads indicate directional movement of organelles. Each panel represents a different organelle type: mitochondria (B), early endosomes (C), recycling endosomes (D), signaling vesicles (E), late endosomes or lysosomes (F), and peroxisomes (G). Panels H to O each include a line graph of calcium ion amplitude over time and a kymograph of organelle movement. Dashed boxes highlight specific time intervals where correlated changes in calcium ion activity and organelle movement occur. The axes of the line graphs remain consistent, with time in seconds on the x-axis and calcium ion amplitude on the y-axis. The kymographs again depict organelle movement over time. Panels H to O correspond to the same organelle types as Panels B to G, allowing comparison of activity patterns across conditions
Ca 2+ oscillation regulates organelle motility in astrocytic processes. (B–F and I–O) Astrocytes co-expressing GCaMP6f and organelle markers: mCherry-Mito for mitochondria (B and I), mCherry-Rab5 for early endosomes (C and J), mCherry-Rab11a for recycling endosomes (D and K), Glut4-mCherry for signaling vesicles (E and L), LAMP1-mCherry for late endosomes/lysosomes (F and M), and PEX3-HaloTag and PEX3-mCherry for peroxisomes (N and O). (A) Schematic of ROI selection in astrocytic processes for simultaneous extraction of Ca2+ amplitude and organelle kymographs. (B–G) Representative examples of organelle dynamics at rest (left) and during global Ca2+ waves (right). For each condition, images show Ca2+ amplitude (left), organelle kymograph from the same ROI (middle), and time-lapse images (right). Magenta arrowheads: directional organelle movements. Scale bar: 10 μm (kymographs), 1 μm (zoom in). Fig. 3 B related to Video 6. (H–O) Temporal alignment of Ca2+ signals (left) and organelle motility (right) from the same ROI over 500 frames acquired at a 0.5-s interval. Dashed boxes highlight periods of correlated changes in Ca2+ activity and organelle movement. Scale bar: 10 μm (kymographs). Fig. 3 I related to Video 7. Fig. 3 J related to Video 8. Fig. 3 K related to Video 9. Fig. 3 L related to Video 10. Fig. 3 M related to Video 11. Fig. 3 N related to Video 12. Fig. 3 O related to Video 13.