In budding yeast, the mitotic spindle moves into the neck between the mother and bud via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. How dynein and microtubules interact with the cortex is unknown. We found that cells lacking Num1p failed to exhibit dynein-dependent microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Num1p localized to the bud cortex, and that localization was independent of microtubules, dynein, or dynactin. These data are consistent with Num1p being an essential element of the cortical attachment mechanism for dynein-dependent sliding of microtubules in the bud.

In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor.

Localization and activation of Elm1 at the bud neck coordinates SPC activity with mother–daughter polarity during cell division.

Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium [1]. More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis [2,3]. Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner. Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes. This may reflect differences in the functions of the actin cytoskeleton in these two organisms. Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S. cerevisiae [4,5] and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin [6]. We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton. The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles. Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant. Taken together, these results suggest that S. cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.

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