Spindle Equator Research Articles

A kinetochore-associated kinesin-7 motor cooperates with BUB3.3 to regulate mitotic chromosome congression in Arabidopsis thaliana.

Faithful genome partition during cell division relies on proper congression of chromosomes to the spindle equator before sister chromatid segregation. Here we uncover a kinesin-7 motor, kinetochore-associated kinesin 1 (KAK1), that is required for mitotic chromosome congression in Arabidopsis. KAK1 associates dynamically with kinetochores throughout mitosis. Loss of KAK1 results in severe defects in chromosome congression at metaphase, yet segregation errors at anaphase are rarely observed. KAK1 specifically interacts with the spindle assembly checkpoint protein BUB3.3 and both proteins show interdependent kinetochore localization. Chromosome misalignment in BUB3.3-depleted plants can be rescued by artificial tethering of KAK1 to kinetochores but not vice versa, demonstrating that KAK1 acts downstream of BUB3.3 to orchestrate microtubule-based chromosome transport at kinetochores. Moreover, we show that KAK1's motor activity is essential for driving chromosome congression to the metaphase plate. Thus, our findings reveal that plants have repurposed BUB3.3 to interface with a specialized kinesin adapted to integrate proper chromosome congression and checkpoint control through a distinct kinetochore design.

Alignment of a Trivalent Chromosome on the Metaphase Plate Is Associated with Differences in Microtubule Density at Each Kinetochore.

Chromosome alignment on the metaphase plate is a conserved phenomenon and is an essential function for correct chromosome segregation for many organisms. Organisms with naturally-occurring trivalent chromosomes provide a useful system for understanding how chromosome alignment is evolutionarily regulated, as they align on the spindle with one kinetochore facing one pole and two facing the opposite pole. We studied chromosome alignment in a praying mantid that has not been previously studied chromosomally, the giant shield mantis Rhombodera megaera. R. megaera has a chromosome number of 2n = 27 in males. Males have X1, X2, and Y chromosomes that combine to form a trivalent in meiosis I. Using live-cell imaging of spermatocytes in meiosis I, we document that sex trivalent Y chromosomes associate with one spindle pole and the two X chromosomes associate with the opposing spindle pole. Sex trivalents congress alongside autosomes, align with them on the metaphase I plate, and then the component chromosomes segregate alongside autosomes in anaphase I. Immunofluorescence imaging and quantification of brightness of kinetochore-microtubule bundles suggest that the X1 and X2 kinetochores are associated with fewer microtubules than the Y kinetochore, likely explaining the alignment of the sex trivalent at the spindle equator with autosomes. These observations in R. megaera support the evolutionary significance of the metaphase alignment of chromosomes and provide part of the explanation for how this alignment is achieved.

Artificial kinetochore beads establish a biorientation-like state in the spindle

Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.

Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells

SummaryChromosome alignment to the spindle equator is a hallmark of mitosis thought to promote chromosome segregation fidelity in metazoans. Yet chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here, we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 125 proteins previously implicated in chromosome alignment. We confirmed chromosome alignment defects upon depletion of 108/125 proteins. Surprisingly, in all confirmed cases, depleted cells frequently entered anaphase after a delay with misaligned chromosomes. Using depletion of prototype proteins resulting in defective chromosome alignment, we show that misaligned chromosomes often satisfy the SAC and directly missegregate without lagging behind in anaphase. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in non-transformed near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.

Chromosome number, sex determination, and meiotic chromosome behavior in the praying mantid Hierodula membranacea.

Praying mantids are important models for studying a wide range of chromosome behaviors, yet few species of mantids have been characterized chromosomally. Here we show that the praying mantid Hierodula membranacea has a chromosome number of 2n = 27, and X1X1X2X2 (female): X1X2Y (male) sex determination. In male meiosis I, the X1, X2, and Y chromosomes of H. membranacea form a sex trivalent, with the Y chromosome associating with one spindle pole and the X1 and X2 chromosomes facing the opposite spindle pole. While it is possible that such a sex trivalent could experience different spindle forces on each side of the trivalent, in H. membranacea the sex trivalent aligns at the spindle equator with all of the autosomes, and then the sex chromosomes separate in anaphase I simultaneously with the autosomes. With this observation, H. membranacea can be used as a model system to study the balance of forces acting on a trivalent during meiosis I and analyze the functional importance of chromosome alignment in metaphase as a preparatory step for subsequent correct chromosome segregation.

Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment.

SummaryChromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment.

The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments: Doing the Right Link.

During mitosis, the interaction of kinetochores (KTs) with microtubules (MTs) drives chromosome congression to the spindle equator and supports the segregation of sister chromatids. Faithful genome partition critically relies on the ability of chromosomes to establish and maintain proper amphitelic end-on attachments, a configuration in which sister KTs are connected to robust MT fibers emanating from opposite spindle poles. Because the capture of spindle MTs by KTs is error prone, cells use mechanisms that sense and correct inaccurate KT-MT interactions before committing to segregate sister chromatids in anaphase. If left unresolved, these errors can result in the unequal distribution of chromosomes and lead to aneuploidy, a hallmark of cancer. In this review, we provide an overview of the molecular strategies that monitor the formation and fine-tuning of KT-MT attachments. We describe the complex network of proteins that operates at the KT-MT interface and discuss how AURORA B and PLK1 coordinate several concurrent events so that the stability of KT-MT attachments is precisely modulated throughout mitotic progression. We also outline updated knowledge on how the RZZ complex is regulated to ensure the formation of end-on attachments and the fidelity of mitosis.

Poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis

ABSTRACT SUMO modification is required for the kinetochore localization of the kinesin-like motor protein CENP-E, which subsequently mediates the alignment of chromosomes to the spindle equator during mitosis. However, the underlying mechanisms by which sumoylation regulates CENP-E kinetochore localization are still unclear. In this study, we first elucidate that the kinetochore protein Nuf2 is not only required for CENP-E kinetochore localization but also preferentially modified by poly-SUMO-2/3 chains. In addition, poly-SUMO-2/3 modification of Nuf2 is significantly upregulated during mitosis, which is temporally correlated to the kinetochore localization of CENP-E during mitosis. We further show that the mitotic defects in CENP-E kinetochore localization and chromosome congression caused by global inhibition of sumoylation can be rescued by expressing a fusion protein between Nuf2 and the SUMO-conjugating enzyme Ubc9 for stimulating Nuf2 SUMO-2/3 modification. Moreover, the expression of another fusion protein between Nuf2 and three SUMO-2 moieties (SUMO-2 trimer), which mimics the trimeric SUMO-2/3 chain modification of Nuf2, can also rescue the mitotic defects due to global inhibition of sumoylation. Conversely, expressing the other forms of Nuf2-SUMO fusion proteins, which imitate Nuf2 modifications by SUMO-2/3 monomer, SUMO-2/3 dimer, and SUMO-1 trimer, respectively, cannot rescue the same mitotic defects. Lastly, compared to Nuf2, the fusion protein simulating the trimeric SUMO-2 chain-modified Nuf2 exhibits a significantly higher binding affinity to CENP-E wild type containing a functional SUMO-interacting motif (SIM) but not the CENP-E SIM mutant. Hence, our results support a model that poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis. Abbreviations: CENP-E, centromere-associated protein E; SUMO, small ubiquitin-related modifier; SIM, SUMO-interacting motif.

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.

Optogenetic Control of Spindle Microtubule Crosslinkers Reveals that Bridging Fibers Promote Chromosome Alignment by Overlap Length-Dependent Forces

During metaphase, chromosome position at the mitotic spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of the microtubule crosslinker PRC1 to disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores.

Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here, we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

Mechanobiology of the Mitotic Spindle.

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances inthe understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements.

Bistability and oscillations in cooperative microtubule and kinetochore dynamics in the mitotic spindle

In the mitotic spindle microtubules attach to kinetochores via catch bonds during metaphase, and microtubule depolymerization forces give rise to stochastic chromosome oscillations. We investigate the cooperative stochastic microtubule dynamics in spindle models consisting of ensembles of parallel microtubules, which attach to a kinetochore via elastic linkers. We include the dynamic instability of microtubules and forces on microtubules and kinetochores from elastic linkers. A one-sided model, where an external force acts on the kinetochore is solved analytically employing a mean-field approach based on Fokker–Planck equations. The solution establishes a bistable force–velocity relation of the microtubule ensemble in agreement with stochastic simulations. We derive constraints on linker stiffness and microtubule number for bistability. The bistable force–velocity relation of the one-sided spindle model gives rise to oscillations in the two-sided model, which can explain stochastic chromosome oscillations in metaphase (directional instability). We derive constraints on linker stiffness and microtubule number for metaphase chromosome oscillations. Including poleward microtubule flux into the model we can provide an explanation for the experimentally observed suppression of chromosome oscillations in cells with high poleward flux velocities. Chromosome oscillations persist in the presence of polar ejection forces, however, with a reduced amplitude and a phase shift between sister kinetochores. Moreover, polar ejection forces are necessary to align the chromosomes at the spindle equator and stabilize an alternating oscillation pattern of the two kinetochores. Finally, we modify the model such that microtubules can only exert tensile forces on the kinetochore resulting in a tug-of-war between the two microtubule ensembles. Then, induced microtubule catastrophes after reaching the kinetochore are necessary to stimulate oscillations. The model can reproduce experimental results for kinetochore oscillations in PtK1 cells quantitatively.

Leaving no-one behind: how CENP-E facilitates chromosome alignment.

Chromosome alignment and biorientation is essential for mitotic progression and genomic stability. Most chromosomes align at the spindle equator in a motor-independent manner. However, a subset of polar kinetochores fail to bi-orient and require a microtubule motor-based transport mechanism to move to the cell equator. Centromere Protein E (CENP-E/KIF10) is a kinesin motor from the Kinesin-7 family, which localizes to unattached kinetochores during mitosis and utilizes plus-end directed microtubule motility to slide mono-oriented chromosomes to the spindle equator. Recent work has revealed how CENP-E cooperates with chromokinesins and dynein to mediate chromosome congression and highlighted its role at aligned chromosomes. Additionally, we have gained new mechanistic insights into the targeting and regulation of CENP-E motor activity at the kinetochore. Here, we will review the function of CENP-E in chromosome congression, the pathways that contribute to CENP-E loading at the kinetochore, and how CENP-E activity is regulated during mitosis.

Spatiotemporal Subcellular Characterization of Absolute NADH Concentration Over the Dynamic Course of Metaphase, Anaphase, Telophase and Cytokinesis using the Phasor Approach to FLIM

Absolute concentration measurements of free and bound nicotinamide adenine dinucleotide hydride (NADH) using the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) is a powerful method for discerning metabolic robustness in living cells with subcellular resolution. Cell cycle and metabolism are at the forefront of ongoing cancer research to explore new targets for cancer therapeutics. A key feature of cancer cells is continual mitotic division. Completion of mitosis ends with the progression of cells through metaphase, anaphase, telophase and cytokinesis. Each of these phases last approximately 3 minutes, presenting a large challenge for researchers to assess metabolism at these stages using available biochemical approaches. Using the phasor FLIM paired with Digital Frequency Domain (DFD) FLIM hardware, we spatiotemporally measure the absolute concentration of NADH (a measure of metabolic robustness) and free/bound NADH ratio (a measure of metabolic mode) in individual mitochondria of cells progressing from metaphase through cytokinesis. Our timelapse FLIM measurements of HeLa cells characterize the metabolic heterogeneity of mitochondria metabolism based on spatial location at the cell equator or spindle poles during the temporal progression of cells from metaphase through cytokinesis. We observe mitochondria NADH concentration and free/bound NADH at the spindle equator and pole correlate in early metaphase, but diverge starting late metaphase to early anaphase coinciding with onset of anaphase cell elongation. Our subcellular NADH FLIM analysis results support the existence of a spatiotemporal specific mechanism for mitochondria function during the course of cell division. Supported by NIGMS P41GM103540 to EG and MD.

Post‐metaphase correction of aberrant kinetochore‐microtubule attachments in mammalian eggs

The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore–microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore–microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore–microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage.

The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes.

Before fertilization, oocytes of most species undergo a long, natural arrest in metaphase. Before this, prometaphase I is also prolonged, due to late stable kinetochore-microtubule attachment. How oocytes stably maintain the dynamic spindle for hours during these periods is poorly understood. Here we report that the bipolar spindle changes its molecular architecture during the long prometaphase/metaphase I in Drosophila melanogaster oocytes. By generating transgenic flies expressing GFP-tagged spindle proteins, we found that 14 of 25 spindle proteins change their distribution in the bipolar spindle. Among them, microtubule cross-linking kinesins, MKlp1/Pavarotti and kinesin-5/Klp61F, accumulate to the spindle equator in late metaphase. We found that the late equator accumulation of MKlp1/Pavarotti is regulated by a mechanism distinct from that in mitosis. While MKlp1/Pavarotti contributes to the control of spindle length, kinesin-5/Klp61F is crucial for maintaining a bipolar spindle during metaphase I arrest. Our study provides novel insight into how oocytes maintain a bipolar spindle during metaphase arrest.

KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle.

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber-binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome Missegregation in Cancer Cell Lines.

For appropriate chromosome segregation, kinetochores on sister chromatids have to attach to microtubules from opposite spindle poles (bi-orientation). Chromosome alignment at the spindle equator, referred to as congression, can occur through the attachment of kinetochores to the lateral surface of spindle microtubules, facilitating bi-orientation establishment. However, the contribution of this phenomenon to mitotic fidelity has not been clarified yet. Here, we addressed whether delayed chromosome alignment to the spindle equator increases the rate of chromosome missegregation. Cancer cell lines depleted of Kid, a chromokinesin involved in chromosome congression, showed chromosome alignment with a slight delay, and increased frequency of lagging chromosomes. Delayed chromosome alignment concomitant with an increased rate of lagging chromosomes was also seen in cells depleted of kinesin family member 4A (KIF4A), another chromokinesin. Cells that underwent chromosome missegregation took relatively longer time to align chromosomes in both control and Kid/KIF4A-depleted cells. Tracking of late-aligning chromosomes showed that they exhibit a higher rate of lagging chromosomes. Intriguingly, the metaphase of cells that underwent chromosome missegregation was shortened, and delaying anaphase onset ameliorated the increased chromosome missegregation. These data suggest that late-aligning chromosomes do not have sufficient time to establish bi-orientation, leading to chromosome missegregation. Our data imply that delayed chromosome alignment is not only a consequence, but also a cause of defective bi-orientation establishment, which can lead to chromosomal instability in cells without severe mitotic defects.

Chromokinesins

During the cell cycle it is critical that the duplicated DNA faithfully segregates to give rise to two genetically identical daughter cells. An even distribution of the genome during mitosis is mediated by mitotic spindle microtubules, assisted by, among others, motor proteins of the kinesin superfamily. Chromokinesins are members of the kinesin superfamily that harbour a specific DNA-binding domain. The best characterized chromokinesins belong to the kinesin-4/Kif4 and kinesin-10/Kif22 families, respectively. Functional analysis of chromokinesins in several model systems revealed their involvement in chromosome arm orientation and oscillations. This is consistent with their originally proposed role in the generation of polar ejection forces that assist chromosome congression to the spindle equator. Kinesin-12/Kif15 members comprise a third family of chromokinesins, but their role remains less understood. Noteworthy, all chromokinesins exhibit chromosome-independent localization on spindle microtubules, and recent works have significantly extended the portfolio of mitotic processes in which chromokinesins play a role, from error correction and DNA compaction, to the regulation of spindle microtubule dynamics.