Pair Of Sister Kinetochores Research Articles

Proximity-based activation of AURORA A by MPS1 potentiates error correction.

Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

A stochastic model for error correction of kinetochore-microtubule attachments in budding yeast.

To divide replicated chromosomes equally between daughter cells, kinetochores must attach to microtubules emanating from opposite poles of the mitotic spindle (biorientation). An error correction mechanism facilitates this process by destabilizing erroneous kinetochore-microtubule attachments. Here we present a stochastic model of kinetochore-microtubule attachments, via an essential protein Ndc80 in budding yeast, Saccharomyces cerevisiae. Using the model, we calculate the stochastic dynamics of a pair of sister kinetochores as they transition among different attachment states. First of all, we determine the kinase-to-phosphatase balance point that maximizes the probability of biorientation, while starting from an erroneous attachment state. We find that the balance point is sensitive to the rates of microtubule-Ndc80 dissociation and derive an approximate analytical formula that defines the balance point. Secondly, we determine the probability of transition from low-tension amphitelic to monotelic attachment and find that, despite this probability being approximately 33%, biorientation can be achieved with high probability. Thirdly, we calculate the contribution of the geometrical orientation of sister kinetochores to the probability of biorientation and show that, in the absence of geometrical orientation, the biorientation error rate is much larger than that observed in experiments. Finally, we study the coupling of the error correction mechanism to the spindle assembly checkpoint by calculating the average binding of checkpoint-related proteins to the kinetochore during the error correction process.

A tension-independent mechanism reduces Aurora B-mediated phosphorylation upon microtubule capture by CENP-E at the kinetochore

ABSTRACTDuring mitosis, Aurora B kinase is required for forming proper bi-oriented kinetochore-microtubule attachments. Current models suggest that tension exerted between a pair of sister-kinetochores (inter-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated microtubule binding substrates, such as the Knl1-Mis12-Ndc80 (KMN) network, resulting in a decrease of phosphorylation and, thus, an increase of affinity for microtubules. Using Single-Molecule High-Resolution Colocalization (SHREC) microscopy analysis of the kinetochore-associated motor CENP-E, we now show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore, and displays a bi-modal Gaussian distribution on a pair of bi-oriented sister kinetochores. The conformational change of CENP-E depends on its microtubule-stimulated motor motility and the highly flexible coiled-coil between its motor and kinetochore-binding tail domains. Chemical inhibition of the motor motility or perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Metaphase chromosome misalignment caused by CENP-E inhibition can be rescued by chemical inhibition of Aurora B kinase. Furthermore, a pair of monotelic sister-kinetochores shows asymmetric levels of Aurora B-mediated phosphorylation in mono-polar spindles depending on CENP-E motor activity. These results collectively suggest a tension-independent mechanism to reduce Aurora B-mediated phosphorylation of outer kinetochore components in response to microtubule capture by CENP-E.

Abstract B27: Kinetochore-microtubule attachments as a precision therapy target

Abstract Glioblastoma multiforme (GBM) is an aggressive and refractory cancer with limited therapeutic strategies and poor survival. One challenge in treating this disease is the ability of single glioblastoma stem-like cells (GSCs) to initiate tumors. To expand the therapeutic repertoire for GBM we performed RNAi screening in GSCs and a proposed cell of origin, neural crest stem cells (NSCs). In these screens we identified multiple regulators of kinetochore-microtubule attachments as specifically required for GSC survival. Kinetochores are composed of hundreds of proteins that form dynamic attachments between microtubules and chromosomes and are essential for genetic fidelity. Antimitotic agents, including those that target kinetochore proteins, are attractive therapeutics because cell division is essential for tumor expansion, but also because mitotic errors can trigger p53-independent apoptotic signals. However, as mitosis is an essential process, most antimitotic drugs have extremely limited therapeutic windows. We demonstrate that specific activities of the mitotic pseudokinase BubR1 are completely dispensable for non-transformed cells, yet become required for survival in a subset of GBM tumors. Through mutational studies we demonstrate that inactivating either BubR1's GLEBS or KARD domain causes lethal chromosome alignment defects in most GSCs and has no observable on-target toxicity in non-transformed cells. We further demonstrate that this cancer-specific requirement arises from oncogenic activation of the MAP kinase pathway, which ultimately hyperactivates Aurora B kinase at kinetochores. Increased phosphorylation of kinetochore proteins weakens their binding to microtubules and can be indirectly measured by a decrease in the distance between sister kinetochore pairs upon microtubule attachment. In limited samples, this measure serves as a functional biomarker for samples that require BubR1 for chromosome alignment. This work has identified multiple precision targets for GBM with dramatic therapeutic windows; moreover, we have initiated development of a biomarker to identify patients that would respond to such a therapy. Citation Format: Jacob A. Herman, Patrick J. Paddison, Jennifer DeLuca, James Olson. Kinetochore-microtubule attachments as a precision therapy target. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B27.

Human kinetochores are swivel joints that mediate microtubule attachments.

Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle - a dynamic microtubule-based force-generating machine. Connections to this spindle are mediated by sister kinetochore pairs, that form dynamic end-on attachments to microtubules emanating from opposite spindle poles. This bi-orientation generates forces that have been reported to stretch the kinetochore itself, which has been suggested to stabilise attachment and silence the spindle checkpoint. We reveal using three dimensional tracking that the outer kinetochore domain can swivel around the inner kinetochore/centromere, which results in large reductions in intra-kinetochore distance (delta) when viewed in lower dimensions. We show that swivel provides a mechanical flexibility that enables kinetochores at the periphery of the spindle to engage microtubules. Swivel reduces as cells approach anaphase, suggesting an organisational change linked to checkpoint satisfaction and/or obligatory changes in kinetochore mechanochemistry may occur before dissolution of sister chromatid cohesion.

Minimal model for collective kinetochore–microtubule dynamics

Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore-MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force-velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.

Nonautonomous Movement of Chromosomes in Mitosis

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence ofkinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores.

Advances in imaging reveal novel insights into the mechanisms promoting accurate chromosome segregation in mitosis and meiosis

The most important outcome of both mitotic and meiotic cell divisions is the accurate segregation of chromosomes. Failure to properly separate sister chromatids during mitosis as well as either homologues or sister chromatids during meiosis I and II, respectively, results in aneuploidy that contributes to tumorigenesis and causes miscarriages, infertility, and birth defects. Speakers at the Mitosis and Meiosis Minisymposium at the ASCB 2010 meeting highlighted new and exciting mechanisms that promote faithful chromosome segregation during these two cell division programs. Duane Compton (Dartmouth Medical School) started the mitosis section by describing recent work using fluorescence dissipation after photoactivation of green fluorescent protein–tagged tubulin to measure the dynamic attachment of microtubules to kinetochores (kMTs). His work showed that molecular changes in the composition of the outer kinetochore generate optimal kMT attachment dynamics during mitotic progression so that kMTs are stable enough to satisfy the spindle assembly checkpoint but not so stable that they prevent the correction of attachment errors. Alexey Khodjakov (Wadsworth Center) showed data using three-dimensional video microscopy that permits him to track the location of single sister kinetochore pairs relative to centrosomes with unprecedented temporal resolution. His work showed how chromosome movements and chromosome arm orientation relative to spindle poles work together to position chromosomes optimally for attachment of kinetochores to spindle microtubules within the brief time window in the early stages of mitosis. Andrew Powers (University of Washington), the recipient of the Norton B. Gilula Award, presented his work using laser trapping to measure the interaction of kinetochores with microtubules. He demonstrated that there is a direct relationship between the force applied to a microtubule and the lifetime that kinetochore complexes hold onto kMT ends, analogous to a Chinese finger trap. The first speaker in the meiosis section was Jennifer Fung (University of California, San Francisco). She described the development of live cell imaging techniques to measure the kinetics of the formation of the synaptonemal complex that assembles between homologous chromosomes during meiosis in most organisms from yeast to humans. Using high-resolution imaging, she showed that, in budding yeast, synapsis proceeds at a rate of ∼30 nm/min and is continuous, with the number of nucleation sites acting as a limiting factor in the speed of this process. Monica Colaiacovo (Harvard Medical School) addressed the poorly understood regulation of establishment and maintenance of sister chromatid cohesion during meiosis in Caenorhabditis elegans. By combining high-resolution imaging and genetic analyses, she demonstrated that LAB-1, a functional analog of Shugoshin, acts to localize cohesin members such as SMC-3 by antagonizing cohesin-destabilizing factors early in prophase, thereby promoting normal progression of chromosome pairing, synapsis, and recombination. Julien Dumont (Institut Curie, Paris) addressed the important question of how chromosomes segregate in the absence of astral microtubules in meiosis. His studies in the context of the C. elegans holocentric chromosomes revealed that microtubules form exclusively between separating chromosomes, suggesting an “inside-out” kinetochore-independent mechanism driving meiotic anaphase chromosome separation. In summary, a theme that emerged from these talks is that improvements in imaging modes and resolution (spatial and temporal) are revealing insights into the mechanisms that drive accurate chromosome segregation in mitosis and meiosis.

Irc15 Is a Microtubule-Associated Protein that Regulates Microtubule Dynamics in Saccharomyces cerevisiae

Microtubules are polymers composed of alpha-beta tubulin heterodimers that assemble into microtubules. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability. Dynamic instability is coupled to the GTPase activity of the beta-tubulin subunit of the tubulin heterodimer. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3). IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11-14]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores.

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.

Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells

We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.

Disruption of Astral Microtubule Contact with the Cell Cortex Activates a Bub1, Bub3, and Mad3-dependent Checkpoint in Fission Yeast

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.

Division of the Nucleolus and Its Release of CDC14 during Anaphase of Meiosis I Depends on Separase, SPO12, and SLK19

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

Attachment of kinetochores to spindle microtubules during meiosis I of Lilium microsporocytes.

Kinetochores and microtubules were visualized simultaneously during spindle formation at the first meiotic division in microsporocytes of Lilium longiflorum (2n = 24) under a confocal laser-scanning microscope, after immunofluorescence staining with centromere-recognizing antiserum and tubulin-specific antibody. During early prometaphase I, each kinetochore of bivalent chromosomes appeared to be an amorphous flat structure upon its initial attachment to microtubules. It became compact and spherical with the development of the spindle. From late prometaphase I, when the bipolar spindle was nearly complete, each kinetochore resembled a double disk that was suggestive of a pair of sister kinetochores and the homologous kinetochores were oriented towards opposite poles. Thus, the bipolar spindle at metaphase I included 12 bivalent chromosomes with a total of four kinetochores each. At anaphase I, the sister kinetochores moved to the same spindle pole as a paired unit. In microsporocytes arrested at prometaphase I by colchicine treatment, the sister kinetochores also came to be distinguishable. These results suggest that the change of kinetochore structure during meiosis I may be under chromosomal control but be somewhat associated with its attachment to spindle microtubules.

The ultrastructure of the diffuse kinetochore in Luzula nivea

Material of Luzula nivea was fixed and processed in the normal way for sectioning and use of the transmission electron microscope. In addition, irradiated as well as control material was employed. Irradiation is known to produce chromosome breaks which rejoin. Apparent side-by-side adherence at metaphase of the chromosomes of Luzula nivea in control material disappeared at the earliest moment of anaphase and because of the high visibility of the kinetochores of this species, one pair of sister kinetochores could be readily associated with one pair of chromatids. Rejoining of chromosome breaks which does not disappear at anaphase, could not be distinguished at metaphase from the above described adherence. Chromatids also were apparently adherent at metaphase and it is pointed out that this is commonly seen in most organisms sectioned for the electron microscope. The apparent adherence is regarded as an artifact of fixation and subsequent processing. The authors do not agree with the interpretation of the chromosomes of Luzula as polycentric. From serial sections it has been established that in Luzula nivea a single diffuse kinetochore extends along most of the length of the chromosome, but does not occupy the whole width.