JCS02419 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Phalloidin-rhodamine labelling of F-actin colocalises extensively with cortical and cytoplasmic structures visualised with transmitted light. Chromaffin cells were incubated with 1 mM phalloidin-rhodamine for 30 minutes at 37°C. In addition to cortical labelling some cells showed cytoplasmic F-actin labelling. Four planes separated by 1 mm of a cell observed under transmitted light (A), or phalloidin-rhodamine confocal fluorescence (B). Extensive colocalisation of cortex and intracellular structures are observed in yellow when both images are merged (C). An image of the mask image with 50% intensity co-labelling of the pixels from the two channels again highlights the high degree of colocalisation. (D). Bar, 1 mm.

• Supplemental Figure 2 -

  Fig. S2. Chemicals affecting F-actin and myosin alter the dynamics of the visualised structures that are unaffected by microtubule inhibitors. Chromaffin cells previously incubated with quinacrine were treated for 15 minutes with F-actin stabilizers (1 mM phalloidin, 10 mM jasplakinolide), dissemblers (5 mM latrunculin A), myosin inhibitors (10 mM BDM, 1 mM wortmannin) or microtubule-affecting chemicals (10 mM vinblastine or 1 mM of taxol labelled with Bodipy 564/570 red fluorescence). After that, high magnification images of epifluorescence and transmitted light channels were obtained at 1 Hz for 1 minute. The images were used to measure X-Y displacement for structures and vesicles. Vesicle and structure mobility in cells treated with BDM (A) and taxol (B). Depicted frames were taken at 10-second intervals. (C) X-Y mobility (mean±s.e.m.) of transmitted light visualised structures under the different experimental conditions. The number of studied cells is also shown. *P<0.01, **P<0.05 compared to mobility levels in the control using Student’s t-test. Bar, 1 mm.

• Supplemental Figure 3 -

  Fig. S3. Stimulation-dependent changes in the transmitted light structures are dependent on the presence of Ca²⁺ in the medium. (A) F-actin cytoskeletal organisation in a cell incubated for 30 seconds with a basal non-stimulating buffer. (B) The same cell after 60 seconds of superfusion with a basal solution containing acetylcholine (10 mM) but lacking calcium (in the presence of 1 mM EGTA). (C) The same cell was incubated for 60 seconds with basal buffer containing 10 mM acetylcholine and 2 mM CaCl₂. Bar, 10 mm.

• Movie 1 -

  Movie 1. Time-lapse study of the mobility of structures visualised by transmitted light scanning microscopy. The movie shows structure movement in rest conditions in a region near the cortical zone at 6´ times speed. The confocal fluorescence channel depicting vesicles has been eliminated from this movie for better appreciation of structure movements. Acquisition at 1 frame per second and played at 6´ acceleration (6 frames per second).

• Movie 2 -

  Movie 2. Time-lapse analysis of the impact of calcium deprivation on the mobility of vesicles and transmitted light structures. Restriction in movement of chromaffin granules (green fluorescence) and transmitted light structures (red LUT) in a cortical region of a cell exposed for 20 minutes to a medium lacking calcium (no added calcium and in the presence of 1 mM EGTA). Acquisition at 1 frame per second and played at 6´ speed (6 frames per second).

• Movie 3 -

  Movie 3. Cytoskeletal changes observed in a chromaffin cell stimulated with acetylcholine. The movie shows the changes observed in the transmitted light channel image corresponding to a chromaffin cell stimulated by perfusion with 10 mM acetylcholine in basal solution. Acquisition at 1 frame per second and played at 6´ speed (6 frames per second).

• Movie 4 -

  Movie 4. Cytoskeletal changes observed in a cortical region from a chromaffin cell stimulated by depolarization. Time-lapse images corresponding to the transmitted light and green fluorescence channels studied in a cortical region from a chromaffin cell stimulated by a transient 10-second superfusion with a high KCl solution (59 mM). Acquisition at 1 frame per second and played at 6´ speed (6 frames per second).