Fig. 7. Effect of ROT1 inactivation on septum formation. (A) Exponentially growing cultures of the tetO₇:ROT1 (JCY216) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Cell were fixed and digested with zymolyase (zym) in the presence of sorbitol. Graph shows the distribution of cells at different cell cycle stages. Values are means+s.d. of three experiments. (B) Exponentially growing cells of the MYO1-GFP (MCY198) strain were synchronized by addition of -factor in the presence of 5 µg/ml doxycycline. DIC images and GFP signal of a collection of cells at the different cell cycle stages are shown. (C,D) Exponentially growing cells of the tetO₇:ROT1 (JCY216) strain were incubated in the presence of 5 µg/ml doxycycline for 10 hours. Cells were fixed and stained with the membrane-specific dye DiI and chitin dye Calcofluor White (CFW) to examine membrane continuity and septum formation in rebudded cells (see text). In C, samples were analyzed by wide-field fluorescence microscopy. A collection of rebudded cells in which a complete septum could not be detected is shown. Picture to the right end shows a control of a rebudded cell with a complete septum. In D, samples were analyzed by confocal microscopy with a series of 140 nm sections. Discontinuous signals were evident in a large fraction of rebudded cells in the case of CFW staining or in all rebudded cells in the case of DiI staining. A selection of the optical sections obtained with one of these cells is shown for each case.