Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Reconstituting the kinetochore–microtubule interface: what, why, and how

The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore–microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule-binding functions of kinetochores in vivo.     

Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Mitosis puts sisters in a strained relationship: force generation at the kinetochore

During mitosis, kinetochores couple chromosomes to the dynamic tips of spindle microtubules. These attachments convert chemical energy stored in the microtubule lattice into mechanical energy, generating force to move chromosomes. In addition to mediating robust microtubule attachments, kinetochores also integrate and respond to regulatory signals that ensure the accuracy of chromosome segregation during each cell division. Signals for corrective detachment act specifically on kinetochore-microtubule attachments that fail to generate normal levels of tension, although it is unclear how tension is sensed and how the attachments are released. In this review, we discuss the mechanisms by which kinetochore-microtubule attachments generate force during chromosome biorientation, and the pathways of maturation and regulation that lead to the formation of correct attachments.     

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.     

Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly

During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.     

Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I

Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore–microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3–4 h, and stabilization of K-MT attachments is delayed an additional 3–4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.     

Stochastic Modelling of Chromosomal Segregation: Errors Can Introduce Correction

Cell division is a complex process requiring the cell to have many internal checks so that division may proceed and be completed correctly. Failure to divide correctly can have serious consequences, including progression to cancer. During mitosis, chromosomal segregation is one such process that is crucial for successful progression. Accurate segregation of chromosomes during mitosis requires regulation of the interactions between chromosomes and spindle microtubules. If left uncorrected, chromosome attachment errors can cause chromosome segregation defects which have serious effects on cell fates. In early prometaphase, where kinetochores are exposed to multiple microtubules originating from the two poles, there are frequent errors in kinetochore-microtubule attachment. Erroneous attachments are classified into two categories, syntelic and merotelic. In this paper, we consider a stochastic model for a possible function of syntelic and merotelic kinetochores, and we provide theoretical evidence that merotely can contribute to lessening the stochastic noise in the time for completion of the mitotic process in eukaryotic cells.     

The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.     

Erroneous Silencing of the Mitotic Checkpoint by Aberrant Spindle Pole-Kinetochore Coordination

To segregate chromosomes during cell division, microtubules that form the bipolar spindle attach to and pull on paired chromosome kinetochores. The spindle assembly checkpoint (SAC) is activated at unattached and misattached kinetochores to prevent further mitotic progression. The SAC is silenced after all the kinetochores establish proper and stable attachment to the spindle. Robust timing of SAC silencing after the last kinetochore-spindle attachment herein dictates the fidelity of chromosome segregation. Chromosome missegregation is rare in typical somatic cell mitosis, but frequent in cancer cell mitosis and in meiosis I of mammalian oocytes. In the latter cases, SAC is normally activated in response to disruptions of kinetochore-spindle attachments, suggesting that frequent chromosome missegregation ensues from faulty SAC silencing. In-depth understanding of how SAC silencing malfunctions in these cases is yet missing, but is believed to hold promise for treatment of cancer and prevention of human miscarriage and birth defects. We previously established a spatiotemporal model that, to the best of our knowledge, explained the robustness of SAC silencing in normal mitosis for the first time. In this article, we take advantage of the whole-cell perspective of the spatiotemporal model to identify possible causes of chromosome missegregation out of the distinct features of spindle assembly exhibited by cancer cells and mammalian oocytes. The model results explain why multipolar spindle could inhibit SAC silencing and spindle pole clustering could promote it—albeit accompanied by more kinetochore attachment errors. The model also eliminates geometric factors as the cause for nonrobust SAC silencing in oocyte meiosis, and instead, suggests atypical kinetochore-spindle attachment in meiosis as a potential culprit. Overall, the model shows that abnormal spindle-pole formation and its aberrant coordination with atypical kinetochore-spindle attachments could compromise the robustness of SAC silencing. Our model highlights systems-level coupling between kinetochore-spindle attachment and spindle-pole formation in SAC silencing.     

The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments

Loss or gain of whole chromosomes, the form of chromosomal instability (CIN) most commonly associated with human cancers, is expected to arise from the failure to accurately segregate chromosomes in mitosis. The mitotic checkpoint is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Another process that prevents errors is stabilization and destabilization of connections between chromosomes and spindle microtubules. An outstanding question is how these two pathways are coordinated to ensure accurate chromosome segregation. Here we show that in human cells depleted of BubR1 - a critical component of the mitotic checkpoint that can directly regulate the onset of anaphase - chromosomes do not form stable attachments to spindle microtubules. Attachments in these cells are restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule attachments. Loss of BubR1 function thus perturbs regulation of attachments rather than the ability of kinetochores to bind to microtubules. Consistent with this finding, depletion of BubR1 increases phosphorylation of CENP-A, a kinetochore-specific Aurora kinase substrate. We propose that BubR1 links regulation of chromosome-spindle attachment to mitotic checkpoint signalling.     

The chromosomal passenger complex activates Polo kinase at centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.     

Loss of KLP-19 polar ejection force causes misorientation and missegregation of holocentric chromosomes

Holocentric chromosomes assemble kinetochores along their length instead of at a focused spot. The elongated expanse of an individual holocentric kinetochore and its potential flexibility heighten the risk of stable attachment to microtubules from both poles of the mitotic spindle (merotelic attachment), and hence aberrant segregation of chromosomes. Little is known about the mechanisms that holocentric species have evolved to avoid this type of error. Our studies of the influence of KLP-19, an essential microtubule motor, on the behavior of holocentric Caenorhabditis elegans chromosomes suggest that it has a major role in combating merotelic attachments. Depletion of KLP-19, which associates with nonkinetochore chromatin, allows aberrant poleward chromosome motion during prometaphase, misalignment of holocentric kinetochores, and multiple anaphase chromosome bridges in all mitotic divisions. Time-lapse movies of GFP-labeled mono- and bipolar spindles demonstrate that KLP-19 generates a force on relatively stiff holocentric chromosomes that pushes them away from poles. We hypothesize that this polar ejection force minimizes merotelic misattachment by maintaining a constant tension on pole-kinetochore connections throughout prometaphase, tension that compels sister kinetochores to face directly toward opposite poles.     

Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes

In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.     

How Do Kinetochores CLASP Dynamic Microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizesto the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

How do kinetochores CLASP dynamic microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

Sensing centromere tension: Aurora B and the regulation of kinetochore function

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.