JCS02762 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Effect of C3 toxin on distribution and morphology of mitochondria. CV-1 cells with mitochondria labeled by EYFP-Mito were microinjected with C3 toxin (1.0 mg/ml) before (A,B) or after transfection with pEGFP-mDia-DN3 (C,D). Fluorescent (A,C) and phase contrast images of the same cells (B,D) were taken 2 hours after microinjection of toxin. Bars, 10 mm.

• Supplemental Figure 2 - Fig. S2. Effects of different agents on actin cytoskeleton in CV-1 cells. Cells were transfected with pEGFP-mDia1-DN3 (A-C and E) or co-transfected with pEGFP-mDia1-DN3 and pEF-BOS-myc-Rho-kinase-RB/PH(TT) (F) and fixed in 4% formaldehyde before (A,E) or after incubation with either 0.4 mM latrunculin B for 20 minutes (B) or with 2.5 mM cytochalasin D for 1 hour (C), or with 30.0 mM Y-27632 for 30 minutes (E). Non-transfected cells were treated with 2.0 mM jasplakinolide (D) for 1 hour and fixed in 4% formaldehyde. F-actin was stained with TRITC-phalloidin. Transfected cells were found before fixation by EGFP fluorescence (shown by arrows). Bar, 10 mm.

• Supplemental Figure 3 - Fig. S3. Effects of F-actin-modifying agents on mitochondria motility in CV-1 cells. Quantification of mitochondria motility in cells expressing EYFP-Mito alone or EYFP-Mito and EGFP-mDia1-DN3 in microinjected cells before or after incubation with either 0.4 mM latrunculin B for 20 minutes, or with 2.5 mM cytochalasin D for 1 hour, or with 2.0 mM jasplakinolide for 1 hour. Values are the mean percentage of fast movements ± s.e.m.; P<0.05; (n = number of cells and in brackets number of organelle movements).

• Movie 1 - Movie 1. Time-lapse recording of CV-1 cell expressing EYFP-Mito. Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 1A.

• Movie 2 - Movie 2. Time-lapse recording of BG2-C2 cells expressing EYFP-Mito. Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 1B.

• Movie 3 - Movie 3. Time-lapse recording of CV-1 cell expressing EYFP-Mito and EGFP-RhoA(Q63L). Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 2E.

• Movie 4 - Movie 4. Time-lapse recording of CV-1 cell expressing EYFP-Mito and EGFP-mDia-DN3. Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 4A.

• Movie 5 - Movie 5. Time-lapse recording of CV-1 cell expressing EYFP-Mito and EGFP-Rac1(Q61L). Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 4B.

• Movie 6 - Movie 6. Time-lapse recording of BG2-C2 cells transfected with pEGFP-C-Dia plasmid (upper cell) and stained with 100 nM Texas Red Mitotracker. Recording rate is 1 frame per 4 seconds, playback rate is 8 frames per second. First frame is shown in Fig. 5I.

• Movie 7 - Movie 7. Time-lapse recording of BG2-C2 cells transfected with pEGFP-C-Dia plasmid (left cell), treated with 10 mM latrunculin B, and stained with Texas Red Mitotracker. (Recording rate is 1 frame per 4 second, playback rate is 8 frames per second). Last frames show green fluorescence in transfected cell and phase contrast image.