Metaphase Spindle Research Articles

Assessment of Spindle Shape Control by Spindle Poleward Flux Measurements and FRAP Bulk Analysis.

In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.

Microtubule organization and plastid distribution during meiosis of Haplomitrium mnioides (Haplomitriopsida)

The organization of microtubules and plastid distribution of the liverwort, Haplomitrium mnioides (Haplomitriopsida), was studied during the meiotic phase lasting for six months. In the late fall, the cytoplasm of early sporocytes forms four lobes of future spore domains before meiotic prophase. Microtubules align at the cytoplasmic cleavage furrow regions as girdling bands in the four-lobed sporocytes. Finally, the cleavage furrows are proximal to the nucleus positioned in the center of the sporocyte, and the girdling bands of the microtubule (GBM) disappear. Subsequently, the nucleus moves into one of the cytoplasmic lobes, and sporocytes pass the winter season at this stage. In early spring, the nucleus returns to the central position of the lobed cytoplasm, concurrent with plastid repositioning around the nucleus. Plastids are then distributed equally to each of the four lobes as a plastid cluster. Astral microtubules emanate from the plastid cluster in each spore domain and encage prophase nuclei as a quadripolar microtubule system (QMS). The QMS changes into a twisted spindle of metaphase I with broad poles, while spindles of metaphase II also emanate from the four plastid clusters. Cytokinesis is completed through the centrifugal cell plate formation in telophase II. The division axes of two successive nuclear divisions appear to be determined by plastid-based QMS, and the future site of cytokinesis is marked by cytoplasmic furrows associated with GBM. The phylogenetic distribution of GBM and QMS suggests that the meiotic system involving these structures is an ancestral trait of liverworts. Long-term dormancy in diploid sporocytes rather than haploid spores may represent transitional traits from charophycean green algae to land plants.

The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis

Faithful chromosome segregation requires bi-oriented kinetochore-microtubule attachment on the metaphase spindle. Aurora B kinase, the catalytic core of the chromosome passage complex (CPC), plays a crucial role in this process. Aurora B activation has widely been investigated in the context of protein phosphorylation. Here, we report that Aurora B is ubiquitinated in mitosis through lysine-63 ubiquitin chains (K63-Ub), which is required for its activation. Mutation of Aurora B at its primary K63 ubiquitin site inhibits its activation, reduces its kinase activity, and disrupts the association of Aurora B with other components of CPC, leading to severe mitotic defects and cell apoptosis. Moreover, we identify that BRCC36 isopeptidase complex (BRISC) is the K63-specific deubiquitinating enzyme for Aurora B. BRISC deficiency augments the accumulation of Aurora B K63-Ubs, leading to Aurora B hyperactivation and erroneous chromosome–microtubule attachments. These findings define the role of K63-linked ubiquitination in regulating Aurora B activation and provide a potential site for Aurora B-targeting drug design.

MCRS1 modulates the heterogeneity of microtubule minus-end morphologies in mitotic spindles.

Faithful chromosome segregation requires the assembly of a bipolar spindle, consisting of two antiparallel microtubule (MT) arrays having most of their minus ends focused at the spindle poles and their plus ends overlapping in the spindle midzone. Spindle assembly, chromosome alignment, and segregation require highly dynamic MTs. The plus ends of MTs have been extensively investigated but their minus-end structure remains poorly characterized. Here, we used large-scale electron tomography to study the morphology of the MT minus ends in three dimensionally reconstructed metaphase spindles in HeLa cells. In contrast to the homogeneous open morphology of the MT plus ends at the kinetochores, we found that MT minus ends are heterogeneous, showing either open or closed morphologies. Silencing the minus end-specific stabilizer, MCRS1 increased the proportion of open MT minus ends. Altogether, these data suggest a correlation between the morphology and the dynamic state of the MT ends. Taking this heterogeneity of the MT minus-end morphologies into account, our work indicates an unsynchronized behavior of MTs at the spindle poles, thus laying the groundwork for further studies on the complexity of MT dynamics regulation.

Mitotic spindle disassembly in human cells relies on CRIPT having hierarchical redox signals.

Swift and complete spindle disassembly in late mitosis is essential for cell survival, yet how it happens is largely unknown in mammalian cells. Here we used real-time live cell microscopy and biochemical assays to show that the primordial dwarfism (PD)-related cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization with a nucleolar concentration during interphase but was distributed to spindles and underwent redox modifications to form disulfide bonds in CXXC pairs during mitosis. Then, it directly interacted with, and might transfer a redox response to, tubulin subunits via a putative redox exchange among cysteine residues to induce microtubule depolymerization. Expression of CRIPT proteins with mutations of these cysteine residues blocked spindle disassembly, generating two cell types with long-lasting metaphase spindles or spindle remnants. Live-cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase might share a common CRIPT-bearing redox-controlled mechanism.

Segregation of the univalent X chromosome in the wide-footed treehopper Enchenopa latipes (Say 1824)

In metaphase I, autosomal bivalents align on the metaphase plate, while naturally-occurring univalent sex chromosomes can display a number of different behaviours depending on cellular conditions. Here we describe the behaviour of the univalent X chromosome in the wide-footed treehopper Enchenopa latipes (Say 1824). We confirm the chromosome number and sex determination method for this species, and that males possess a univalent X chromosome. We show that the univalent X chromosome forms a bipolar attachment to the spindle in metaphase I, and then segregates intact toward one spindle pole in late anaphase I (long after autosomes have initiated poleward movement). Movement of the univalent toward one pole is associated with loss of microtubule connections toward the opposite spindle pole.

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex.1,2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse+TIPs that control microtubule dynamics and interactions with other intracellular structures.3 Because binding of many+TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated+TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of+TIP association with growing microtubule ends in live cells.7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

Spindle and Kinetochore-associated Family Genes are Prognostic and Predictive Biomarkers in Hepatocellular Carcinoma.

Background and AimsHepatocellular carcinoma (HCC) is one of the most frequent malignant tumors. Spindle and kinetochore-associated (SKA) family genes are essential for the maintenance of the metaphase plate and spindle checkpoint silencing during mitosis. Recent studies have indicated that dysregulation of SKA family genes induces tumorigenesis, tumor progression, and chemoresistance via modulation of cell cycle and DNA replication. However, the differential transcription of SKAs in the context of HCC and its prognostic significance has not been demonstrated.MethodsBioinformatics analyses were performed using TCGA, ONCOMINE, HCCDB, Kaplan-Meier plotter, STRING, GEPIA databases. qRT-PCR, western blot, and functional assays were utilized for in vitro experiments.ResultsWe found remarkable upregulation of transcripts of SKA family genes in HCC samples compared with normal liver samples on bioinformatics analyses and in vitro validation. Interaction analysis and enrichment analysis showed that SKA family members were mainly related to microtubule motor activity, mitosis, and cell cycle. Immuno-infiltration analysis showed a correlation of all SKA family genes with various immune cell subsets, especially T helper 2 (Th2) cells. Transcriptional levels of SKA family members were positively associated with histologic grade, T stage, and α-fetoprotein in HCC patients. Receiver operating characteristic curve analysis demonstrated a strong predictive ability of SKA1/2/3 for HCC. Increased expression of these SKAs was associated with unfavorable overall survival, progression-free survival, and disease-specific survival. On Cox proportional hazards regression analyses, SKA1 upregulation and pathological staging were independent predictors of overall survival and disease-specific survival of HCC patients. Finally, clinical tissue microarray validation and in vitro functional assays revealed SKA1 acts an important regulatory role in tumor malignant behavior.ConclusionsSKA family members may potentially serve as diagnostic and prognostic markers in the context of HCC. The correlation between SKAs and immune cell infiltration provides a promising research direction for SKA-targeted immunotherapeutics for HCC.

Centromere Tension Measurement in Budding Yeast Mitosis.

During budding yeast mitosis, duplicated chromosomes are aligned at the center of the metaphase mitotic spindle, and the centromeres are stretched by forces generated within the mitotic spindle. In response to these stretching forces, mechanical tension builds up in the centromeric chromatin. The magnitude of this tension is detected by the cell to signal the attachment configuration of the sister chromosomes: a high tension signal would indicate that sister chromosomes are properly attached to opposite spindle poles, while a low tension signal could indicate the lack of a bipolar attachment. A low tension signal drives the cell to correct improper attachments in metaphase, thus preventing potential errors in anaphase chromosome segregation. In this paper, we describe a microscopy-based method to directly measure the magnitude of centromere tension in budding yeast metaphase spindles. The advantage of this method is that quantitative tension estimates are obtained without perturbing spindle and/or chromosome structure and as cells progress normally through mitosis.

Altered microfilament dynamics contribute to the formation of diploid metaphase spindles in porcine oocytes which fail to reach the metaphase-II stage during in vitro maturation.

Premature meiotic arrest during in vitro maturation (IVM) of porcine oocytes after germinal vesicle breakdown is associated with microfilament degradation. We aimed to clarify (1) if such arrest occurs at the metaphase-I (MI) stage or the oocyte progresses to a so-called diploid metaphase-II (MII) stage and (2) if microfilament degradation is the cause or result of the meiotic arrest. The number and morphology of chromosomes in oocytes showing premature meiotic arrest at 44 h IVM (38 monovalents) was similar to those cultured in the presence of the actin polymerization-inhibitor cytochalasin-B, but different from those of MI-stage (19 bivalents), and MII-stage oocytes (19 monovalents) at 33 and 44 h of IVM, respectively. Immunostaining revealed similar frequencies of microfilament degradation in prematurely arrested and cytochalasin-B-treated oocytes (58.7% and 57.2%, respectively), which were higher (P < 0.05) than those in MI- and MII-stage oocytes (10.6% and 6.8%, respectively). Induction of MI-arrest by nocodazole did not affect microfilament morphology. ATP and mRNA levels of microfilament-related genes in oocytes were similar among all groups. These results suggest that altered microfilament dynamics contribute to the formation of diploid metaphase spindles in oocytes, which fail to reach the MII stage. However, the cause of microfilament degeneration remains unclear.

Ginsenoside Rg1 suppresses cancer cell proliferation through perturbing mitotic progression

BackgroundAlthough the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methodsCancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. ResultsGinsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. ConclusionGinsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

Slk19 enhances cross-linking of microtubules by Ase1 and Stu1.

The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19’s localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.

Ctf3/CENP-I provides a docking site for the desumoylase Ulp2 at the kinetochore.

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

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.

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.

Evidence of Non-microtubule Spindle Forces in Mesostoma ehrenbergii Spermatocytes.

We tested conclusions reached in previous experiments in which Mesostoma spermatocyte chromosomes moved rapidly to a pole in the absence of microtubules: after 10 μM nocodazole (NOC) depolymerized metaphase spindle microtubules, kinetochores from each of the 3 bivalents detached from the same pole and rapidly moved to the other pole, at speeds averaging 37.7 μm/min. with some as high as 100 μm/min. We concluded that these very fast movements were due to non-microtubule forces arising from a spindle matrix. However, since the chromosomes stretch out before detaching, there is tension in the chromosomes from the stretch. Thus the movements of detached kinetochores conceivably might be due to recoil from the tension, though we argued against this possibility (Fegaras and Forer, 2018a). In this article we test whether recoil causes the movements. We cut bivalents into 2 pieces, using a femtosecond laser, before addition of NOC. When 1 bivalent was severed, all kinetochores moved to one pole in 12/15 cells; when 2 bivalents were severed, all kinetochores moved to one pole in 4/6 cells; and when all 3 bivalents were severed all kinetochores moved to one pole in 3/9 cells. The bivalent “halves” moved rapidly, with average speeds of 47 μm/min, velocities that are not significantly different from those in cells without any laser-cut bivalents (p > 0.05). Since kinetochores move at the same speeds whether they are part of bivalents or not, NOC-induced chromosome movements are not due to recoil from tension along the full-length bivalent, strongly supporting the idea that non-microtubule forces move chromosomes in Mesostoma spermatocytes.

KINESIN-12E regulates metaphase spindle flux and helps control spindle size in Arabidopsis.

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.

The EB1-Kinesin-14 complex is required for efficient metaphase spindle assembly and kinetochore bi-orientation.

Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1-Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1-Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1-Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.

Klp2 and Ase1 synergize to maintain meiotic spindle stability during metaphase I

The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule cross-linker Ase1, as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.

Microtubules Enhance Mesoscale Effective Diffusivity in the Crowded Metaphase Cytoplasm.

Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by ~30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments.