Slk19 enhances cross-linking of microtubules by Ase1 and Stu1.

The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19's localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.

Unattached kinetochores drive their own capturing by sequestering a CLASP.

Kinetochores that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. We show that they also promote their own capturing. Similar to what governs the localization of spindle assembly checkpoint proteins, the phosphorylation of Spc105 by Mps1 allows unattached kinetochores to sequester Stu1 in cooperation with Slk19. The withdrawal of Stu1, a CLASP essential for spindle integrity, from microtubules and attached kinetochores disrupts the organization of the spindle and thus allows the enhanced formation of dynamic random microtubules that span the nucleus and are ideal to capture unattached kinetochores. The enhanced formation of nuclear random microtubules does not occur if Stu1 sequestering to unattached kinetochores fails and the spindle remains uncompromised. Consequently, these cells exhibit a severely decreased capturing efficiency. After the capturing event, Stu1 is relocated to the capturing microtubule and prevents precocious microtubule depolymerization as long as kinetochores are laterally or incompletely end-on attached.