Microtubule nucleation is spatiotemporally regulated in cells by several known molecules, including the template γ-tubulin and the polymerase XMAP215. The role of XMAP215 in nucleation is under debate, specifically whether it acts independently as a polymerase or acts dependently with γ-tubulin. We first confirm XMAP215 as a classically defined nucleator that reduces the nucleation lag seen in bulk tubulin assembly. Secondly, using deletion constructs, we probe the domain requirements for XMAP215 to promote microtubule nucleation. We show that its ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of tumor overexpressed gene (TOG) domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct. These findings suggest there are at least two independent processes in nucleation, one promoted by γ-tubulin and one promoted by XMAP215. We propose that XMAP215 accelerates the addition of subunits to existing nucleation intermediates formed either spontaneously or by oligomers of γ-tubulin. [Media: see text].

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.

Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid comigration in diverse organisms, but direct evidence for such fusion has been lacking. We used laser trapping and quantitative fluorescence microscopy to study native kinetochore particles isolated from yeast. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I-specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid comigration, a conserved feature of meiosis that is fundamental to Mendelian inheritance.