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. 2012 Oct 1;23(19):3814–3826. doi: 10.1091/mbc.E12-05-0400

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© 2012 Chen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — BNI1 is essential in rewired cells. (A) Synthetic lethality of bni1 BEM1-SNC2. The spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY12648 and 12778). (B–E) Rewired cells carrying a temperature-sensitive Bni1p (DLY12226, compared with the control DLY8601) progress through the cell cycle but are defective in polarization and budding. G1-phase cells were enriched by centrifugal elutriation at 4°C and released into 37°C media to resume growth. Samples were fixed and scored for percentage budded (n > 100) (B) and percentage cells with S or G2 DNA content (n = 10,000) (C). (D) DIC images of cells released into 37°C media for 160 min. Most cells did not bud, and those that did formed very wide necks and did not complete cytokinesis. (E) Inverted time-lapse images of G1-phase cells, which were released and filmed at 37°C. The control BEM1-GFP-SNC2 cell (top) started to polarize and bud, whereas a similar-sized BEM1-GFP-SNC2 bni1-12 cell (bottom) grew big and round without establishing polarity. Scale bar, 5 μm.