Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells

1. Download : Download high-res image (232KB)
2. Download : Download full-size image

     

CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore‐microtubule dynamics to promote mitotic progression and fidelity

Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore–microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b–CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin–CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1–astrin–Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.     

Characterization of plant Aurora kinases during mitosis

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.†These authors equally contributed to this work     

TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes

Chromosomal instability can arise from defects in chromosome–microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1''s role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome–microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore–microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.     

The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment

Faithful chromosome segregation relies on dynamic interactions between spindle microtubules and chromosomes. Especially, all chromosomes must be aligned at the equator of the spindle to establish bi-orientation before they start to segregate. The spindle assembly checkpoint (SAC) monitors this process, inhibiting chromosome segregation until all chromosomes achieve bi-orientation. The original concept of ‘checkpoints’ was proposed as an external surveillance system that does not play an active role in the process it monitors. However, accumulating evidence from recent studies suggests that SAC components do play an active role in chromosome bi-orientation. In this review, we highlight a novel Mad1 role in chromosome alignment, which is the first conserved mechanism that links the SAC and kinesin-mediated chromosome gliding.     

Reverse engineering of force integration during mitosis in the Drosophila embryo

The mitotic spindle is a complex macromolecular machine that coordinates accurate chromosome segregation. The spindle accomplishes its function using forces generated by microtubules (MTs) and multiple molecular motors, but how these forces are integrated remains unclear, since the temporal activation profiles and the mechanical characteristics of the relevant motors are largely unknown. Here, we developed a computational search algorithm that uses experimental measurements to ‘reverse engineer’ molecular mechanical machines. Our algorithm uses measurements of length time series for wild‐type and experimentally perturbed spindles to identify mechanistic models for coordination of the mitotic force generators in Drosophila embryo spindles. The search eliminated thousands of possible models and identified six distinct strategies for MT–motor integration that agree with available data. Many features of these six predicted strategies are conserved, including a persistent kinesin‐5‐driven sliding filament mechanism combined with the anaphase B‐specific inhibition of a kinesin‐13 MT depolymerase on spindle poles. Such conserved features allow predictions of force–velocity characteristics and activation–deactivation profiles of key mitotic motors. Identified differences among the six predicted strategies regarding the mechanisms of prometaphase and anaphase spindle elongation suggest future experiments.     

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.     

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2-terminal coiled-coil domain with the capacity for self assembly. On the basis of these findings, we propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss‐of‐function screenings with analysis of MT‐dynamics in human cells to investigate the molecular mechanisms underlying MT‐flux. We report that kinesin‐7/CENP‐E at kinetochores (KTs) is the predominant driver of MT‐flux in early prometaphase, while kinesin‐4/KIF4A on chromosome arms facilitates MT‐flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin‐5/EG5 and kinesin‐12/KIF15, and our data suggest that the MT‐flux driving force is transmitted from non‐KT‐MTs to KT‐MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT‐flux rate correlates with spindle length, and this correlation depends on the establishment of stable end‐on KT‐MT attachments. Strikingly, we find that MT‐flux is required to regulate spindle length by counteracting kinesin 13/MCAK‐dependent MT‐depolymerization. Thus, our study unveils the long‐sought mechanism of MT‐flux in human cells as relying on the coordinated action of four kinesins to compensate for MT‐depolymerization and regulate spindle length.     

癌症高表达蛋白--Hec1在纺锤体组装检查点中的作用

细胞增殖依赖于细胞分裂前染色体的复制及随后的姐妹染色单体分离到达两极。纺锤体组装检查点具有确保染色体信息传递保真性的作用,检查点的缺失可能导致染色体的分离异常和肿瘤形成。癌症高表达蛋白(Hec1)通过与调控G2/M期的蛋白间的相互作用而在染色体的分离中发挥重要作用。Hec1与Nuf2的复合物,在G2/M期与动粒相结合,Hec1的缺失将导致严重的染色体分离错误。Hec1具有召集Mps1和Mad1/Mad2复合物结合到动粒上的作用,这种结合可以激活纺锤体组装检查点途径中非常重要的APCCdc20途径。但是Hec1、Mps1、Mad1三者之间的相互作用仍未明了。Hec1还可以通过与26S蛋白酶复合物的不同亚基结合调控其功能。Hec1是一种丝氨酸磷酸化蛋白,其磷酸化是由Nek2在G2/M期完成的。     

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis

Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

染色体乘客复合体对细胞有丝分裂的调控作用

染色体乘客复合体(CPC)是近年来处于细胞有丝分裂调控研究热点的一组蛋白分子,主要由Aurora B激酶、着丝粒中心蛋白(INCENP)、Survivin及Borealin/DasarB等蛋白分子组成。研究证实CPC在有丝分裂过程中扮演了重要的角色,涉及纺锤体形成、染色体排列、姊妹染色单体分离、纺锤体检查点信号及胞质分裂等多种重要功能的调节。本文重点阐述了CPC各组成蛋白功能特点、相互作用及对纺锤体检查点蛋白和微管蛋白的调节等方面的最新研究进展,同时阐明CPC组成蛋白作为抗癌药物研制靶标的潜在应用价值。     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Exotic mitotic mechanisms

The emergence of eukaryotes around two billion years ago provided new challenges for the chromosome segregation machineries: the physical separation of multiple large and linear chromosomes from the microtubule-organizing centres by the nuclear envelope. In this review, we set out the diverse solutions that eukaryotic cells use to solve this problem, and show how stepping away from ‘mainstream’ mitosis can teach us much about the mechanisms and mechanics that can drive chromosome segregation. We discuss the evidence for a close functional and physical relationship between membranes, nuclear pores and kinetochores in generating the forces necessary for chromosome segregation during mitosis.