John C. Meadows

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint.

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.

The DASH complex and Klp5/Klp6 kinesin coordinate bipolar chromosome attachment in fission yeast

We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13‐117 (cyclin B) and mal3‐1 (EB‐1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere‐associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment.

Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors.

The spindle checkpoint is the prime cell-cycle control mechanism that ensures sister chromatids are bioriented before anaphase takes place. Aurora B kinase, the catalytic subunit of the chromosome passenger complex, both destabilizes kinetochore attachments that do not generate tension and simultaneously maintains the spindle checkpoint signal. However, it is unclear how the checkpoint is silenced following chromosome biorientation. We demonstrate that association of type 1 phosphatase (PP1(Dis2)) with both the N terminus of Spc7 and the nonmotor domains of the Klp5-Klp6 (kinesin-8) complex is necessary to counteract Aurora B kinase to efficiently silence the spindle checkpoint. The role of Klp5 and Klp6 in checkpoint silencing is specific to this class of kinesin and independent of their motor activities. These data demonstrate that at least two distinct pools of PP1, one kinetochore associated and the other motor associated, are needed to silence the spindle checkpoint.

The Dam1/DASH complex is required for the retrieval of unclustered kinetochores in fission yeast

In fission yeast centromeres cluster at the nuclear envelope in a region underlying the spindle pole body during interphase, an arrangement known as a Rabl configuration. We have identified a strain in which one pair of sister kinetochores is unclustered from the others and binds the nuclear envelope at a point distal to the spindle pole body. We show that during mitosis unclustered kinetochores are captured by intranuclear spindle microtubules which then pull the kinetochores back to one of the two spindle poles before they are bi-oriented on the mitotic spindle. We find that kinetochore retrieval occurs at the depolymerising microtubule plus end and is dependent on the non-essential Dam1/DASH complex. In the absence of Dam1 unclustered kinetochores are captured on the lateral surface of spindle microtubule bundles but poleward kinetochore movement does not occur. These data provide the first direct evidence that the Dam1/DASH complex can couple the force generated by microtubule depolymerisation to direct chromosome movement in vivo.

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.

Latrunculin A delays anaphase onset in fission yeast by disrupting an Ase1-independent pathway controlling mitotic spindle stability.

It has been proposed previously that latrunculin A, an inhibitor of actin polymerization, delays the onset of anaphase by causing spindle misorientation in fission yeast. However, we show that Delta mto1 cells, which are defective in nucleation of cytoplasmic microtubules, have profoundly misoriented spindles but are not delayed in the timing of sister chromatid separation, providing compelling evidence that fission yeast does not possess a spindle orientation checkpoint. Instead, we show that latrunculin A delays anaphase onset by disrupting interpolar microtubule stability. This effect is abolished in a latrunculin A-insensitive actin mutant and exacerbated in cells lacking Ase1, which cross-links antiparallel interpolar microtubules at the spindle midzone both before and after anaphase. These data indicate that both Ase1 and an intact actin cytoskeleton are required for preanaphase spindle stability. Finally, we show that loss of Ase1 activates a checkpoint that requires only the Mad3, Bub1, and Mph1, but not Mad1, Mad2, or Bub3 checkpoint proteins.

Interplay between mitotic kinesins and the Aurora kinase-PP1 (protein phosphatase 1) axis.

Correct transmission of genetic information from mother to daughter cells is necessary for development and survival. Accurate segregation is achieved by bipolar attachment of sister kinetochores in each chromatid pair to spindle microtubules emanating from opposite spindle poles, a process known as chromosome bi-orientation. Achieving this requires dynamic interplay between kinetochore proteins, kinesin motor proteins and cell cycle regulators. Chromosome bi-orientation is monitored by a surveillance mechanism known as the SAC (spindle assembly checkpoint). The Aurora B kinase, which is bound to the inner centromere during early mitosis, plays a central role in both chromosome bi-orientation and the spindle checkpoint. The application of tension across centromeres establishes a spatial gradient of high phosphorylation activity at the inner centromere and low phosphorylation activity at the outer kinetochore. This gradient is further refined by the association of PP1 (protein phosphatase 1) to the outer kinetochore, which stabilizes kinetochore-microtubule interactions and silences the spindle checkpoint by dephosphorylating Aurora B kinase targets when chromosome bi-orientation is achieved. In the present review, I discuss emerging evidence that bidirectional cross-talk between mitotic kinesins and the Aurora kinase-PP1 axis is crucial for co-ordinating chromosome bi-orientation and spindle checkpoint signalling in eukaryotes.

Plo1 phosphorylates Dam1 to promote chromosome bi-orientation in fission yeast

The fungal-specific heterodecameric outer kinetochore DASH complex facilitates the interaction of kinetochores with spindle microtubules. In budding yeast, where kinetochores bind a single microtubule, the DASH complex is essential, and phosphorylation of Dam1 by the Aurora kinase homologue, Ipl1, causes detachment of kinetochores from spindle microtubules. We demonstrate that in the distantly related fission yeast, where the DASH complex is not essential for viability and kinetochores bind multiple microtubules, Dam1 is instead phosphorylated on serine 143 by the Polo kinase homologue, Plo1, during prometaphase and metaphase. This phosphorylation site is conserved in most fungal Dam1 proteins, including budding yeast Dam1. We show that Dam1 phosphorylation by Plo1 is dispensable for DASH assembly and chromosome retrieval but instead aids tension-dependent chromosome bi-orientation.

Identification of a Sgo2-dependent but Mad2-independent pathway controlling anaphase onset in fission yeast

Summary The onset of anaphase is triggered by activation of the anaphase-promoting complex/cyclosome (APC/C) following silencing of the spindle assembly checkpoint (SAC). APC/C triggers ubiquitination of Securin and Cyclin B, which leads to loss of sister chromatid cohesion and inactivation of Cyclin B/Cdk1, respectively. This promotes relocalization of Aurora B kinase and other components of the chromosome passenger complex (CPC) from centromeres to the spindle midzone. In fission yeast, this is mediated by Clp1 phosphatase-dependent interaction of CPC with Klp9/MKLP2 (kinesin-6). When this interaction is disrupted, kinetochores bi-orient normally, but APC/C activation is delayed via a mechanism that requires Sgo2 and some (Bub1, Mph1/Mps1, and Mad3), but not all (Mad1 and Mad2), components of the SAC and the first, but not second, lysine, glutamic acid, glutamine (KEN) box in Mad3. These data indicate that interaction of CPC with Klp9 terminates a Sgo2-dependent, but Mad2-independent, APC/C-inhibitory pathway that is distinct from the canonical SAC.

Cell biology: polar expeditions for PP1.

A new study shows that phospho-dependent expulsion of type-1-phosphatase (PP1) from the spindle pole by Fin1 (NIMA) kinase ensures switch-like activation of Cyclin B-Cdk1 at the G2/M transition.