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. 2021 Jul 15;32(15):1374–1392. doi: 10.1091/mbc.E20-11-0699

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© 2021 Kishimoto et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 9: — Overexpression of Sfk1 excludes GFPenvy-D4H from the mother cell PM. (A) The polarized distribution of GFPenvy-D4H. Wild-type or sfk1∆ cells carrying pRS316-GFPenvy-D4H were grown in SD-Ura medium to mid–log phase at 30°C. “Polarized,” “partially polarized,” and “not polarized” localizations of GFPenvy-D4H are indicated with pink, yellow, and green arrows, respectively. Right panel illustrates the budding yeast cell cycle. Three patterns of the GFPenvy-D4H localization are shown. (B) Complementary localization of GFPenvy-D4H and Sfk1-3xmCherry to daughter (bud) and mother cells, respectively. Wild-type cells expressing these proteins were grown at 30°C. To show endogenously expressed Sfk1-3xmCherry clearly, the brightness was adjusted to make it brighter. (C) Fluorescence intensity profile of a cell showing the “polarized” pattern of GFPenvy-D4H. Fluorescence signals were quantified along the dotted line from the mother cell to the bud. The brightness of Sfk1-3xmCherry was adjusted as in B. (D) Quantification of three GFPenvy-D4H localization patterns. The cells in A were examined. The percentage of cells showing “polarized,” “partially polarized,” and “not polarized” localizations of GFPenvy-D4H was determined as described in Materials and Methods and is expressed as the mean ± SD of three independent experiments (n > 150 cells in total for each strain). “n.s.” indicates no significant difference between all combinations as determined by a two-tailed Student’s t test. (E) Heterogeneous (high and low) expression of Sfk1-mCherry by a multicopy plasmid. Wild-type cells carrying pRS316-GFPenvy-D4H and YEplac181-SFK1-mCherry were grown in SD-Leu-Ura medium to mid–log phase at 30°C. The brightness was not adjusted after background subtraction. Arrows indicate cells highly (yellow) and lowly (cyan) expressing Sfk1-mCherry. (F) High expression of Sfk1-mCherry significantly increased the “polarized” pattern of GFPenvy-D4H. Cells were examined and categorized as in D. Low or high expression of Sfk1-mCherry was determined as described in the legend of Supplemental Figure 10B. Bars: No, control plasmid; Endo, endogenous expression of Sfk1-3xmCherry; Low, multicopy plasmid of SFK1-mCherry but low expression of Sfk1-mCherry; High, multicopy plasmid of SFK1-mCherry and high expression of Sfk1-mCherry. The percentage of cells showing the indicated patterns is expressed as the mean ± SD of three independent experiments (n > 103 cells in total for each strain). An asterisk indicates a significant difference, as determined by the Tukey–Kramer test (p < 0.05), in the “polarized” and “not polarized” patterns. (G) GFPenvy-D4H was exclusively distributed to the bud in a cell highly expressing Sfk1-mCherry. The brightness is not adjusted after background subtraction. The right panel represents the fluorescence intensity profile quantified as in C. Bars, 3 µm.