Figure 7.
IMM diffusion barriers exist constitutively and OMM diffusion barriers are formed in response to stresses. (A and B) Dual-color FLIP in fis1∆ cells expressing Yta12-GFP (IMM protein) and matrix-mCherry. Representative images and pooled quantification of Yta12-GFP FLIP and t70 of Yta12-GFP are shown. Cells grown in YPD medium (A; n = 23 cells; n ≥ 6 cells for each experiment). Cells grown in YPE (B; n = 25 cells; n ≥ 5 cells for each experiment). (C) Compartmentalization indexes (tX) are calculated as tX (time to reduce to X% of the total fluorescence) in the bud (tX [B]) compared with tX in the mother (tX [M]) (upper panel). Compartmentalization indexes (t70) of Yta12-GFP FLIP data from A and B (lower panel). Welch’s two-tailed t test was applied to compare the tX in the mother and bud. (D) GFP FLIP in fis1∆ cells expressing Alo1-GFP (OMM protein) in the absence of matrix-mCherry. Representative images and pooled quantification of Alo1-GFP FLIP are shown (n = 23 cells). (E and F) Dual-color FLIP in fis1∆ cells expressing Tom20-GFP and mCherry fused to the transmembrane region of Tom20 (Tom20TM-mCherry). Representative images and line graphs of the intensity profiles along the indicated lines (E). Quantification of Tom20-GFP and Tom20TM-mCherry (F; n = 17 cells). (G) Dual-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) and matrix-mCherry grown in YPD medium (n = 30 cells). (H) Compartmentalization indexes (t50) were calculated from Alo1-GFP (OMM protein) FLIP in fis1∆ cells grown on YPD, YPG, YPE, or YPDE in the presence or absence of matrix-mCherry. Data from at least three independent experiments are shown. n ≥ 5 cells for each clone, and a total of 42 cells (YPD without mCherry), 34 cells (YPG without mCherry), 54 cells (YPD with mCherry), 49 cells (YPE with mCherry), or 25 cells (YPDE with mCherry) were analyzed. Compartmentalization indexes (t50) were compared by one-way ANOVA followed by the Tukey’s method. (I) Single-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) in the absence of matrix-mCherry grown in YPG medium (n = 34 cells). (J) Dual-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) and matrix-mCherry grown in YPE medium (n = 49 cells). Photobleach was applied 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 ± SE (hereafter, shadowed error bars in the FLIP experiments are changed to SE to compare the means among groups instead of showing distributions within a group). Error bar: mean ± SE.

IMM diffusion barriers exist constitutively and OMM diffusion barriers are formed in response to stresses. (A and B) Dual-color FLIP in fis1∆ cells expressing Yta12-GFP (IMM protein) and matrix-mCherry. Representative images and pooled quantification of Yta12-GFP FLIP and t70 of Yta12-GFP are shown. Cells grown in YPD medium (A; n = 23 cells; n ≥ 6 cells for each experiment). Cells grown in YPE (B; n = 25 cells; n ≥ 5 cells for each experiment). (C) Compartmentalization indexes (tX) are calculated as tX (time to reduce to X% of the total fluorescence) in the bud (tX [B]) compared with tX in the mother (tX [M]) (upper panel). Compartmentalization indexes (t70) of Yta12-GFP FLIP data from A and B (lower panel). Welch’s two-tailed t test was applied to compare the tX in the mother and bud. (D) GFP FLIP in fis1∆ cells expressing Alo1-GFP (OMM protein) in the absence of matrix-mCherry. Representative images and pooled quantification of Alo1-GFP FLIP are shown (n = 23 cells). (E and F) Dual-color FLIP in fis1∆ cells expressing Tom20-GFP and mCherry fused to the transmembrane region of Tom20 (Tom20TM-mCherry). Representative images and line graphs of the intensity profiles along the indicated lines (E). Quantification of Tom20-GFP and Tom20TM-mCherry (F; n = 17 cells). (G) Dual-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) and matrix-mCherry grown in YPD medium (n = 30 cells). (H) Compartmentalization indexes (t50) were calculated from Alo1-GFP (OMM protein) FLIP in fis1∆ cells grown on YPD, YPG, YPE, or YPDE in the presence or absence of matrix-mCherry. Data from at least three independent experiments are shown. n ≥ 5 cells for each clone, and a total of 42 cells (YPD without mCherry), 34 cells (YPG without mCherry), 54 cells (YPD with mCherry), 49 cells (YPE with mCherry), or 25 cells (YPDE with mCherry) were analyzed. Compartmentalization indexes (t50) were compared by one-way ANOVA followed by the Tukey’s method. (I) Single-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) in the absence of matrix-mCherry grown in YPG medium (n = 34 cells). (J) Dual-color FLIP in fis1∆ cells expressing Alo1-GFP (OMM) and matrix-mCherry grown in YPE medium (n = 49 cells). Photobleach was applied 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 ± SE (hereafter, shadowed error bars in the FLIP experiments are changed to SE to compare the means among groups instead of showing distributions within a group). Error bar: mean ± SE.

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