Bud6 Directs Sequential Microtubule Interactions with the Bud Tip and Bud Neck during Spindle Morphogenesis inSaccharomyces cerevisiae

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Abstract
In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule–cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule–neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Δmutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule–cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.