Cells with tethered centromeres can activate the spindle checkpoint.

<p>(<b>A</b>) To assay spindle checkpoint activation of cells in the presence of microtubule-depolymerizing drugs, cells were synchronized in G1 with alpha factor, and released into media containing microtubule-depolymerizing drugs (benomyl and nocodazole). Samples were collected every 60 minutes and scored for the large-budded phenotype indicative of checkpoint activation. (<b>B</b>) The ability of both control (dimeric) and tethered (tetrameric) cells to activate the spindle checkpoint was assayed by scoring the percent of cells arrested as large-budded cells, and compared to cells that do not possess a functional checkpoint and cannot arrest (<i>mad2Δ</i>). Cells expressing either the dimeric or tetrameric Lac repressor arrested in the drugs, indicating that the spindle checkpoint was functional in both strains. 200 cells scored for large-budded phenotype; error bars represent the standard deviation of 3 independent trials. (<b>C</b>) To assay the ability of cells to recover from spindle checkpoint activation induced by microtubule-depolymerizing drugs, cells were synchronized in G1 with alpha factor, released into media containing benomyl and nocodazole for 90 minutes then washed and transferred to media without drugs. Samples were collected every 10 minutes and scored for anaphase (GFP dots in both mother and daughter cells) (<b>D</b>) The ability of both control and tethered strains to recover from spindle checkpoint activation was assayed by transiently treating cells with microtubule-depolymerizing drugs. Both strains arrested in the presence of the drugs and did not enter anaphase until after the drugs were washed out at T = 90 minutes (red arrow). 100 cells were scored for anaphase for each strain in each experiment; error bars represent the standard deviation of at least 3 independent trials.