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Fig. 6. Bub2p affects cell cycle progression and morphogenesis in dynein-depleted yeast cells. (A) Immunoblots of PMET3-GFP-Dyn1p expression (left) or repression (right). Strains YJB6755 (PMET3-GFP-CDC3/CDC3, lanes 1 and 4), YJB7944 (DYN1-GFP/DYN1, lanes 2 and 5) and YJB8046 (PMET3-GFP-DYN1/dyn1
, lanes 3 and 6) were grown overnight in PMET3 ON conditions, diluted to OD600=0.01 in fresh SDC medium containing 10 mM methionine and 2 mM cysteine (PMET3 OFF) or lacking methionine and cysteine (PMET3 ON) and incubated at 30°C for 14 hours. Immunoblots were probed with antibodies against GFP or histone H4, as described in Materials and Methods. Dyn1-GFP in YJB7944 was not detectable under these conditions, presumably owing to low expression from the native promoter; it was detectable by microscopy (supplementary material Fig. S1). (B) Bub2p monitors two steps of anaphase in dynein-depleted cells. The strains indicated were grown as in A. The number of DAPI-stained nuclei per cell is plotted as a histogram at the side of each micrograph. Strains: wild-type, BWP17; bub2/, CB432; PMET3-DYN1/dyn1, YJB8039; PMET3-DYN1/dyn1 bub2/, YJB9993. (C) Bub2p inhibits disassembly of the mitotic spindle in dynein-deficient cells. Strain YJB10232 (PMET3-DYN1/ bub2/ TUB2-GFP:NAT1) was cultured as in A. When PMET3-DYN1 was expressed (left), mitotic spindles remained intact until after the dSPB penetrated the bud; when PMET3-DYN1 was repressed (right), mitotic spindles disassembled prematurely. DIC and fluorescent images were merged in B and C. Bars, 5 µm.
