A mitochondrial diffusion barrier exists at the bud neck. (A) Two possible scenarios upon change of photobleaching areas from the mother (upper images) to bud (lower images). In the presence of a diffusion barrier at the bud neck, indicated by the red disk, bleaching in the bud would result in reversed bleaching curves (left). Slower diffusion in the bud compartment (absence of a diffusion barrier) would result in both compartments losing fluorescence in a similar manner upon bleaching in the bud (right). (B) Example images, pooled quantification of GFP FLIP, and t₇₀ of GFP FLIP in fis1∆ cells expressing Tom20-GFP and matrix-mCherry (n = 34 cells, n ≥ 10 cells for each experiment). Photobleach was applied in the GFP and mCherry channels in the bud as indicated by white circles. Images are a sum projection of five z-stacks taken at 0.5-μm intervals. Scale bar: 3 μm. Shadows represent mean ± SD. Error bar: mean ± SE. Welch’s two-tailed t test was applied to compare the t_X in the mother and bud.