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

Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5+ and klp6+ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A

Bub1 and BubR1: at the Interface between Chromosome Attachment and the Spindle Checkpoint ▿

The spindle checkpoint ensures genome fidelity by temporarily halting chromosome segregation and the ensuing mitotic exit until the last kinetochore is productively attached to the mitotic spindle. At the interface between proper chromosome attachment and the metaphase-to-anaphase transition are the mammalian spindle checkpoint kinases. Compelling evidence indicates that the checkpoint kinases Bub1 and BubR1 have the added task of regulating kinetochore-microtubule attachments. However, the debate on the requirement of kinase activity is in full swing. This minireview summarizes recent advances in our understanding of the core spindle checkpoint kinases Bub1 and BubR1 and considers evidence that supports and opposes the role of kinase activity in regulating their functions during mitosis

The Aspergillus nidulans Kinesin-3 UncA Motor Moves Vesicles along a Subpopulation of Microtubules

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncArigor decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncArigor but reacted with a specific antibody against tyrosinated α-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules