The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).

A minimal mathematical model based on stochastic attachment and detachment of kinetochore microtubules accurately reproduces both normal and abnormal chromosome segregation in fission yeast.

The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stressactivated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/ Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

The tug of war that ensues when a kinetochore binds microtubules from both spindle poles is resolved by Ase1/Prc1.

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place. INTRODUCTIONIn eukaryotes, the separation of sister chromatids is mediated by the interaction of spindle microtubules with specialized regions of chromosomes known as kinetochores. At the start of prometaphase, kinetochores are not attached to microtubules. The kinetochore of one sister chromatid then captures a microtubule nucleated from a spindle pole. Once its sister kinetochore has captured microtubules from the other pole, the chromosome becomes bioriented. During metaphase, all bioriented chromosomes move to the equatorial plane, known in animal cells as the metaphase plate, in a process called chromosome congression (Rieder and Salmon, 1994). At anaphase, cohesion is lost, allowing sister chromatids to separate to their respective poles. Finally, the cytokinetic actomyosin ring contracts perpendicularly to, and at the site of, the spindle midzone to ensure that each set of sister chromatids is separated to daughter cells.Cell cycle progression in all eukaryotic cells is monitored by a series of checkpoints that ensure both the fidelity and the temporal and spatial order of cell cycle events (Hartwell and Weinert, 1989). One of the best studied of these is a checkpoint that monitors the assembly of the mitotic spindle (Yu, 2002;Zhou et al., 2002;Cleveland et al., 2003). Components of this checkpoint, which is often referred to as the spindle assembly checkpoint (SAC), were first identified in budding yeast and include the Mad1, Mad2, Mad3, Bub1, Bub3, and Mps1 proteins (Li and Murray, 1991;Hoyt et al., 1991;Weiss and Winey, 1996). Structural and functional homologues of these proteins have been identified in all other eukaryotes so far examined, including fission yeast (He et al., 19...

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end-directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB). We find that the minus-end-directed motor Klp2 colocalizes with the kinetochore during its transport to the SPB and contributes to the effectiveness of retrieval by affecting both end-on pulling and lateral sliding. Furthermore, we provide in vivo evidence that Dam1, a component of the DASH complex, also colocalizes with the kinetochore during its transport and is essential for its retrieval by either of these mechanisms. Finally, we find that the position of the unattached kinetochore correlates with the size and orientation of the INMs, suggesting that chromosome recapture may not be a random process.

In animal cells, the mitotic spindle is aligned perpendicular to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring (CAR). We show that, in fission yeast, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton. Interaction initially occurs with a region surrounding the nucleus, which we term the astral microtubule interaction zone (AMIZ). Simultaneous contact of astral microtubules from both poles with the AMIZ directs spindle rotation and this requires both actin and two type V myosins, Myo51 and Myo52. Astral microtubules from one pole only then contact the CAR, which is located at the centre of the AMIZ. We demonstrate that the anillin homologue Mid1, which dictates correct placement of the CAR, is necessary to stabilise the mitotic spindle perpendicular to the axis of cell division. Finally, we show that the position of the mitotic spindle is monitored by a checkpoint that regulates the timing of sister chromatid separation.

In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. In higher eukaryotes, kinesin-8 family members control chromosome congression by regulating their oscillations. By contrast, here, we demonstrate that fission yeast kinesin-8 controls chromosome congression by an alternative mechanism. We propose that kinesin-8 aligns chromosomes by controlling pulling forces in a length-dependent manner. A coarse-grained model of chromosome segregation implemented with a length-dependent process that controls the force at kinetochores is necessary and sufficient to mimic kinetochore alignment, and prevents the appearance of lagging chromosomes. Taken together, these data illustrate how the local action of a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes and to prevent aneuploidy.