Bub3p facilitates spindle checkpoint silencing in fission yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3 Delta cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest

Shugoshin Promotes Sister Kinetochore Biorientation in Saccharomyces cerevisiae

Chromosome segregation must be executed accurately during both mitotic and meiotic cell divisions. Sgo1 plays a key role in ensuring faithful chromosome segregation in at least two ways. During meiosis this protein regulates the removal of cohesins, the proteins that hold sister chromatids together, from chromosomes. During mitosis, Sgo1 is required for sensing the absence of tension caused by sister kinetochores not being attached to microtubules emanating from opposite poles. Here we describe a differential requirement for Sgo1 in the segregation of homologous chromosomes and sister chromatids. Sgo1 plays only a minor role in segregating homologous chromosomes at meiosis I. In contrast, Sgo1 is important to bias sister kinetochores toward biorientation. We suggest that Sgo1 acts at sister kinetochores to promote their biorientation

Shugoshin 2 Regulates Localization of the Chromosomal Passenger Proteins in Fission Yeast Mitosis

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells

Unrestrained spindle elongation during recovery from spindle checkpoint activation in cdc15-2 cells results in mis-segregation of chromosomes

10.1091/mbc.E09-07-0637Molecular Biology of the Cell21142384-2398MBCE

Function and Assembly of DNA Looping, Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis