Compartmentalization in mitochondria tethered to the cell poles. (A–F) Dual-color FLIP in fis1∆ cells expressing GFP-tagged mitochondrial proteins and matrix-mCherry. (A and B) Representative images of fis1∆ cells expressing Atp1-GFP and matrix-targeted mCherry with mitochondrial accumulation both at the mother and bud poles (A) or only at the bud pole (B). Atp1-GFP shows delayed bleaching in the tethered mitochondrial masses whereas matrix-mCherry does not. (C and D) Representative images of fis1∆ cells expressing Hem1-GFP and matrix-targeted mCherry with mitochondrial accumulation both at the mother and bud poles (C) or only at the bud pole (D). Soluble matrix proteins do not show delayed bleaching in the tethered mitochondrial masses. (E and F) Representative images (E) and GFP quantification (F) of fis1∆ cells expressing Tom20-GFP and matrix-targeted mCherry after cytokinesis. Mitochondrial masses that are tethered to the former bud pole-side of daughter cells (Pole; n = 15 cells) show a delayed diffusion pattern of Tom20-GFP whereas those located at the former bud neck side (Neck; n = 15 cells) do not. Light shadows represent mean ± SD and dark shadows represent mean ± SE. Photobleach was applied in the GFP and mCherry channels as indicated by white circles. Images are a sum projection of five z-stacks taken at 0.5-μm intervals. Scale bar: 3 μm. 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.