Regulation of mitosis by kinetochore-dependent mechanisms
Final Report Abstract
In S. cerevisiae, unattached kinetochores sequester the microtubule (MT) rescue factor Stu1 and Slk19. The abduction of Stu1 from MTs compromises the pro/metaphase spindle and thus allows the formation of dynamic capturing microtubules that search the nucleus. How Slk19 sequestering compromises metaphase spindles is much less clear. Upon kinetochore capturing the sequestered proteins are deposited onto the capturing MTs and subsequently move to the spindle pole. Based on this we addressed three questions: (1) Can we reconstitute the sequestering of Stul and Slk19 ectopically and learn more about its regulation and minimal requirements? Similar to activation of the spindle assembly checkpoint, sequestering of Stu1 and Slk19 at unattached kinetochores is triggered by Mps1 dependent phosphorylation of the kinetochore protein Spc105. We localized a Spc105 mutant that is defective for kinetochore localization to ectopic chromosomal sites and induced its interaction with the protein kinase Mps1. This most likely activated the spindle assembly checkpoint, but it did not trigger any Stu1 / Slk19 sequestering. Thus phosphorylation of Spc105 by Mps1 may not be sufficient to start the sequestering process. (2) How does Slk19 support the formation of metaphase spindles? We identified a Slk19 deletion mutant that failed to localize to the spindle but still localized to kinetochores and applied this mutant to investigate Slk19’s spindle function. We showed that metaphase spindle localization of Slk19 depends on Ase1 (MT crosslinker) and Stu1. Vice versa, Slk19 strongly enhanced the localization of Ase1 and Stu1 to metaphase spindles in vivo and at MTs formed in vitro. Furthermore Slk19 also enhanced the crosslinking of MTs by Ase1 and Stu1 in vitro. Consistently, defective MT localization of Slk19 resulted in a spindle phenotype indicative of compromised MT alignment. We thus suggest that Slk19 amplifies the localization of Ase1 and Stu1 at metaphase spindles and thus promotes and stabiles MT overlaps. (3) Does an active removal of MT stabilizing proteins from not aligned MTs contribute to spindle formation? We found that the removal of Stul from capturing MTs depends mostly on the motor activity of Vik1/Kar3. Interfering with it maintained capturing MTs with Stu1 long after the kinetochores had reached the spindle region. Furthermore it promoted the formation of not-aligned MTs in metaphase (with kinetochores completely attached). In both cases, this correlated with defective spindles. The not aligned MTs not only harbored Stul, but also in large quantities Asel and to a smaller extent Stu2. Not aligned MTs formed in the presence of functional Vikl/Kar3 carried much less of these proteins. Furthermore we found that Vikl/Kar3 can transport Stul and Asel in vitro and that the interaction with Vikl/Kar3 depends on the MT binding regions of both proteins. We therefore suggest that Vikl/Kar3 removes MT-stabilizing protein from not aligned MTs. This allows depolymerization and/or alignment of these MTs and thus facilitates spindle formation.
Publications
- (2018). Unattached kinetochores drive their own capturing by sequestering a CLASP. Nat Commun 9, 886
Kolenda, C., Ortiz, J., Pelzl, M., Norell, S., Schmeiser, V., and Lechner, J.
(See online at https://doi.org/10.1038/s41467-018-03108-z) - (2021). Slk19 enhances cross-linking of microtubules by Ase1 and Stu1. Mol Biol Cell 32, ar22
Norell, S., Ortiz, J., and Lechner, J.
(See online at https://doi.org/10.1091/mbc.e21-05-0279)
