Figure 6.
Mitochondrial masses tethered at cell poles are compartmentalized. (A–H) Dual-color FLIP in fis1∆ cells expressing GFP-tagged mitochondrial proteins and matrix-mCherry, categorized according to their morphologies. Example images (A–D), pooled quantification of GFP FLIP, and t70 of GFP FLIP from three or four independent experiments (E–H). Shadows represent mean ± SD. (A and E) Cells with mitochondrial accumulation both at the mother and bud poles (E; n = 40 cells, n = 10 cells for each experiment). (B and F) Cells with mitochondrial accumulation only at the bud pole (F; n = 28 cells, n ≥ 9 cells for each experiment). (C and G) Cells with mitochondrial accumulation only at the mother pole (G; n = 39 cells, n ≥ 10 cells for each experiment). (D and H) Cells without mitochondrial accumulation at the poles (H; n = 29 cells, n ≥ 9 cells for each experiment). Arrows indicate mitochondrial accumulation at cell poles. Welch’s two-tailed t test was applied to compare the tX in the mother and bud. (I) t70 (bud) from E-H are compared among the morphology groups. t70 (bud) values in each category were compared by one-way ANOVA followed by Tukey’s method. (J) FLIP was applied in the mitochondrial tubules at 10 pxs away from the mitochondrial masses as illustrated in the model (left). Representative images of FLIP experiments in cells with mitochondrial masses without (left, indicated by an arrow) or with (right, indicated by a double arrow) attachment at the mother cell poles. (K) The pooled mother bleaching curves in cells that have mitochondrial masses with (n = 23 cells) or without (n = 24 cells) attachment at the mother cell poles. Light shadows represent mean ± SD and dark shadows represent mean ± SE. Data were fitted to the nonlinear regression curves and analyzed based on a null hypothesis “one curve for all data sets” and an alternative hypothesis of “different curve for each data set” using the extra sum-of-squares F-test. (L) FRAP was performed within the mitochondrial masses with (n = 35 cells) or without (n = 36 cells) attachment at the mother cell poles. Tom20-GFP fluorescence within the bleached area was obtained and normalized to the average of the final 200 data points (frame numbers 401–600; 100%). Shadows represent mean ± SD. Photobleaching was applied in GFP and mCherry channels as indicated with white circles. Images are a sum projection of five z-stacks taken at 0.5-μm intervals. Scale bar: 3 μm. Error bar: mean ± SE.

Mitochondrial masses tethered at cell poles are compartmentalized. (A–H) Dual-color FLIP in fis1∆ cells expressing GFP-tagged mitochondrial proteins and matrix-mCherry, categorized according to their morphologies. Example images (A–D), pooled quantification of GFP FLIP, and t70 of GFP FLIP from three or four independent experiments (E–H). Shadows represent mean ± SD. (A and E) Cells with mitochondrial accumulation both at the mother and bud poles (E; n = 40 cells, n = 10 cells for each experiment). (B and F) Cells with mitochondrial accumulation only at the bud pole (F; n = 28 cells, n ≥ 9 cells for each experiment). (C and G) Cells with mitochondrial accumulation only at the mother pole (G; n = 39 cells, n ≥ 10 cells for each experiment). (D and H) Cells without mitochondrial accumulation at the poles (H; n = 29 cells, n ≥ 9 cells for each experiment). Arrows indicate mitochondrial accumulation at cell poles. Welch’s two-tailed t test was applied to compare the tX in the mother and bud. (I) t70 (bud) from E-H are compared among the morphology groups. t70 (bud) values in each category were compared by one-way ANOVA followed by Tukey’s method. (J) FLIP was applied in the mitochondrial tubules at 10 pxs away from the mitochondrial masses as illustrated in the model (left). Representative images of FLIP experiments in cells with mitochondrial masses without (left, indicated by an arrow) or with (right, indicated by a double arrow) attachment at the mother cell poles. (K) The pooled mother bleaching curves in cells that have mitochondrial masses with (n = 23 cells) or without (n = 24 cells) attachment at the mother cell poles. Light shadows represent mean ± SD and dark shadows represent mean ± SE. Data were fitted to the nonlinear regression curves and analyzed based on a null hypothesis “one curve for all data sets” and an alternative hypothesis of “different curve for each data set” using the extra sum-of-squares F-test. (L) FRAP was performed within the mitochondrial masses with (n = 35 cells) or without (n = 36 cells) attachment at the mother cell poles. Tom20-GFP fluorescence within the bleached area was obtained and normalized to the average of the final 200 data points (frame numbers 401–600; 100%). Shadows represent mean ± SD. Photobleaching was applied in GFP and mCherry channels as indicated with white circles. Images are a sum projection of five z-stacks taken at 0.5-μm intervals. Scale bar: 3 μm. Error bar: mean ± SE.

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