FIGURE 3:

Bni1p regulation is essential in rewired cells. (A) A catalytic fragment of Bni1p (FH1–FH2) can provide formin function in wild-type but not rewired cells. Full-length Bni1p or the FH1–FH2 fragment was tested for the ability to rescue temperature-sensitive formin function in rewired (bni1-12 BEM1-SNC2; DLY12226, 14024, 12620) and otherwise wild-type (bni1-116 bnr1; DLY7924, 14025, 12627) strains at 37°C. Serial dilutions of cell cultures (containing ∼10⁴, 10³, 10², and 10¹ cells) were spotted on plates with rich media and incubated at the indicated temperature for 2.5 d. (B) FH1–FH2 does not rescue the synthetic lethality of bni1 BEM1-SNC2. Spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY12848, 14272). (C) FH1–FH2 is not dominantly deleterious in rewired cells. DIC images of exponentially growing rewired cells expressing (right; DLY14811) or not expressing (left; DLY13095) FH1–FH2 in addition to full-length Bni1p. Images were assembled from multiple fields (denoted with black boxes). Scale bar, 5 μm. (D) Immunoblotting shows expression of the full-length Bni1p (DLY14024) and the FH1-FH2 fragment (DLY12620, 12848, 14272) in the strains used in A and B. Cdc11p (septin) serves as loading control.