Metaphase Spindle Research Articles

Marking and Measuring Single Microtubules by PRC1 and Kinesin-4

Error-free cell division depends on the assembly of the spindle midzone, a specialized array of overlapping microtubules that emerges between segregating chromosomes during anaphase. The molecular mechanisms by which a subset of dynamic microtubules from the metaphase spindle are selected and organized into a stable midzone array are poorly understood. Here, we show using in vitro reconstitution assays that PRC1 and kinesin-4, two microtubule-associated proteins required for midzone assembly, can tag microtubule plus ends. Remarkably, the size of these tags is proportional to filament length. We determine the crystal structure of the PRC1 homodimer and map the protein-protein interactions needed for tagging microtubule ends. Importantly, length-dependent microtubule plus-end-tagging by PRC1 is also observed in dividing cells. Our findings suggest how biochemically similar microtubules can be differentially marked, based on length, for selective regulation during the formation of specialized arrays, such as those required for cytokinesis.

Augmin-dependent microtubule nucleation at microtubule walls in the spindle

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end-wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.

Dynamical Scenarios for Chromosome Bi-orientation

Chromosome bi-orientation at the metaphase spindle is essential for precise segregation of the genetic material. The process is error-prone, and error-correction mechanisms exist to switch misaligned chromosomes to the correct, bi-oriented configuration. Here, we analyze several possible dynamical scenarios to explore how cells might achieve correct bi-orientation in an efficient and robust manner. We first illustrate that tension-mediated feedback between the sister kinetochores can give rise to a bistable switch, which allows robust distinction between a loose attachment with low tension and a strong attachment with high tension. However, this mechanism has difficulties in explaining how bi-orientation is initiated starting from unattached kinetochores. We propose four possible mechanisms to overcome this problem (exploiting molecular noise; allowing an efficient attachment of kinetochores already in the absence of tension; a trial-and-error oscillation; and a stochastic bistable switch), and assess their impact on the bi-orientation process. Based on our results and supported by experimental data, we put forward a trial-and-error oscillation and a stochastic bistable switch as two elegant mechanisms with the potential to promote bi-orientation both efficiently and robustly.

A checkpoint escape

In most dividing cells, the spindle assembly checkpoint (SAC), which monitors kinetochore–microtubule interactions, signals to prevent anaphase onset until all chromosomes are correctly attached to the metaphase spindle. Here, Shao et al. reveal that Xenopus laevis oocytes do not respond to a SAC. The authors first developed a karyotyping technique that allows the analysis of meiotic chromosomes after nuclear envelop breakdown. When treating oocytes during meiosis I with microtubule-depolymerizing drugs that destroyed the spindle, they observed that homologous chromosomes could still divide into single pairs of chromatids, and on time. This suggested that oocytes could progress to meiosis II and arrest at metaphase II, as normal, without a spindle. Similarly, defective oocytes exhibiting monopolar spindles proceeded through monopolar anaphase without delay, indicating that, in X. laevis, the metaphase-to-anaphase transition is not under SAC regulation.

Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

Rab24 is Required for Normal Cell Division

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.

An allometric relationship between mitotic spindle width, spindle length, and ploidy inCaenorhabditis elegansembryos

The mitotic spindle is a diamond-shaped molecular apparatus crucial for chromosomal segregation. The regulation of spindle length is well studied, but little is known about spindle width. Previous studies suggested that the spindle can self-organize to maintain a constant aspect ratio between its length and width against physical perturbations. Here we determine the widths of metaphase spindles of various sizes observed during embryogenesis in Caenorhabditis elegans, including small spindles obtained by knocking down the tpxl-1 or spd-2 gene. The spindle width correlates well with the spindle length, but the aspect ratio between the spindle length and spindle width is not constant, indicating an allometric relationship between these parameters. We characterize how DNA quantity (ploidy) affects spindle shape by using haploid and polyploid embryos. We find that the length of the hypotenuse, which corresponds to the distance from the apex of the metaphase plate to the spindle pole, remains constant in each cell stage, regardless of ploidy. On the basis of the quantitative data, we deduce an allometric equation that describes the spindle width as a function of the length of the hypotenuse and ploidy. On the basis of this equation, we propose a force-balance model to determine the spindle width.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis albaL.) seedlings

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 μg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 μg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Physical Aspects of Spindle Assembly

The spindle is a complex assembly of microtubules, motors, and other associated proteins, which segregate chromosomes during cell division.In metaphase, the spindle exists in a steady-state with a constant flux of molecules and energy continuously modifying and maintaining its architecture. While the self-organization of systems of microtubules and motors have been investigated using theory and experiments, there have been few attempts to test if the proposed theories can be used to understand the dynamics and structure of complex biological systems in vivo. Here we use polarized light microscopy, 3D time-lapse spinning disk confocal microscopy, single molecule imaging, second harmonic generation microscopy, and mechanical measurements to test the validity of continuum models of metaphase spindles. Our results show that a simple continuum model can quantitatively explain spindle structure and dynamics, demonstrate that rigorous physical theories can be used to quantitatively describe complex subcellular systems, and provides a framework for understanding the structure of the spindle and its response to physical and molecular perturbations.

Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

4D imaging reveals a shift in chromosome segregation dynamics during mouse pre-implantation development

Cells of the early developing mammalian embryo frequently mis-segregate chromosomes during cell division, causing daughter cells to inherit an erroneous numbers of chromosomes. Why the embryo is so susceptible to errors is unknown, and the mechanisms that embryos employ to accomplish chromosome segregation are poorly understood. Chromosome segregation is performed by the spindle, a fusiform-shaped microtubule-based transient organelle. Here we present a detailed analysis of 4D fluorescence-confocal data sets of live embryos progressing from the one-cell embryo stage through to blastocyst in vitro, providing some of the first mechanistic insights into chromosome segregation in the mammalian embryo. We show that chromosome segregation occurs as a combined result of poleward chromosome motion (anaphase-A) and spindle elongation (anaphase-B), which occur simultaneously at the time of cell division. Unexpectedly, however, regulation of the two anaphase mechanisms changes significantly between the first and second embryonic mitoses. In one-cell embryos, the velocity of anaphase-A chromosome motion and the velocity and overall extent of anaphase-B spindle elongation are significantly constrained compared with later stages. As a result chromosomes are delivered close to the center of the forming two-cell stage blastomeres at the end of the first mitosis. In subsequent divisions, anaphase-B spindle elongation is faster and more extensive, resulting in the delivery of chromosomes to the distal plasma membrane of the newly forming blastomeres. Metaphase spindle length scales with cell size from the two-cell stage onwards, but is substantially shorter in the first mitosis than in the second mitosis, and the duration of mitosis-1 is substantially greater than subsequent divisions. Thus, there is a striking and unexpected shift in the approach to cell division between the first and second mitotic divisions, which likely reflects adaptations to the unique environment within the developing embryo.

Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation.

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore-microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B-dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gα(i/o) during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gα(i)·GDP. The crystal structures of Gα(i)·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gα(i) interaction features when compared with the only high resolution structure known with GL/Gα(i) interaction between RGS14 and Gα(i1.) Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gα(i)·GDP. A highly conserved "double Arg finger" sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gα(i). Together with the sequence alignment, we suggest that the LGN GL/Gα(i) interaction represents a general binding mode between GL motifs and Gα(i). We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors.

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Chromosome positioning at the equator of the mitotic spindle emerges out of a relatively entropic background. At this moment, termed metaphase, all kinetochores have typically captured microtubules leading to satisfaction of the spindle-assembly checkpoint, but the cell does not enter anaphase immediately. The waiting time in metaphase is related to the kinetics of securin and cyclin B1 degradation, which trigger sister-chromatid separation and promote anaphase processivity, respectively. Yet, as judged by metaphase duration, such kinetics vary widely between cell types and organisms, with no evident correlation to ploidy or cell size. During metaphase, many animal and plant spindles are also characterized by a conspicuous "flux" activity characterized by continuous poleward translocation of spindle microtubules, which maintain steady-state length and position. Whether spindle microtubule flux plays a specific role during metaphase remains arguable. Based on known experimental parameters, we have performed a comparative analysis amongst different cell types from different organisms and show that spindle length, metaphase duration and flux velocity combine within each system to obey a quasi-universal rule. As so, knowledge of two of these parameters is enough to estimate the third. This trend indicates that metaphase duration is tuned to allow approximately one kinetochore-to-pole round of microtubule flux. We propose that the time cells spend in metaphase evolved as a quality enhancement step that allows for the uniform stabilization/correction of kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

Analyzing Fission Yeast Multidrug Resistance Mechanisms to Develop a Genetically Tractable Model System for Chemical Biology

Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a "closed" mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

P21-activated kinase 4 regulates mitotic spindle positioning and orientation

During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex.1 Spindle orientation is also regulated by integrin-mediated cell adhesion2 and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell.3 Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial.4 Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division.5 In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation.

Tension-dependent nucleosome remodeling at the pericentromere in yeast

Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

To explain how micrometer-sized cellular structures generate and respond to forces, we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based setup to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multimode imaging system allows the tracking of microtubules, chromosomes and needle tips. This setup can be used to analyze the viscoelastic properties of the spindle on timescales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 d.

Spatiotemporal Regulation of Ipl1/Aurora Activity by Direct Cdk1 Phosphorylation

Oscillating cyclin-dependent kinase 1 (Cdk1) activity is the major regulator of cell-cycle progression, whereas the Aurora B kinase, as part of the chromosome passenger complex (CPC), controls critical aspects of mitosis such as chromosome condensation and biorientation on the spindle. How these kinases mechanistically coordinate their important functions is only partially understood. Here, using budding yeast, we identify a regulatory mechanism by which the Cdk1 kinase Cdc28 directly controls the Aurora kinase Ipl1. We show that Cdk1 phosphorylates Ipl1 on two serine residues in the N-terminal domain, thereby suppressing its association with the microtubule plus-end tracking protein Bim1 until the onset of anaphase. Failure to phosphorylate Ipl1 leads to its premature targeting to the metaphase spindle and results in constitutive Bim1 phosphorylation, which is normally restricted to anaphase. Cells expressing an Ipl1-Sli15 complex that cannot be phosphorylated by Cdk1 display a severe growth defect. Our work shows that Ipl1/Aurora is not only the catalytic subunit of the CPC but also an important regulatory target that allows Cdk1 to coordinate chromosome biorientation with spindle morphogenesis.