Stu1 inversely regulates kinetochore capture and spindle stability.

The Saccharomyces cerevisiae CLASP (CLIP-associated protein) Stu1 is essential for the establishment and maintenance of the mitotic spindle. Furthermore, Stu1 localizes to kinetochores. Here we show that, in prometaphase, Stu1 assembles in an Ndc80-dependent manner exclusively at kinetochores that are not attached to microtubules. Stu1 relocates to microtubules when a captured kinetochore reaches a spindle pole. This relocation does not depend on kinetochore biorientation, but requires a functional DASH complex. Stu1 at detached kinetochores facilitates kinetochore capturing. Furthermore, since most of the nuclear Stu1 is sequestered by one or a few detached kinetochores, the presence of detached kinetochores prevents Stu1 from localizing the spindle, and therefore from stabilizing the spindle. Thus, the sequestering of Stu1 by detached kinetochores serves as a checkpoint that keeps spindle poles in close proximity until all kinetochores are captured. This is likely to facilitate kinetochore biorientation. In agreement with the findings described above, a kinetochore mutant (okp1-52) that fails to release Stu1 from the kinetochore displays a severe spindle defect upon spindle pole body separation, and this defect can be rescued by destroying the okp1-52 kinetochore.