Understanding the underlying mechanisms governing spindle orientation: How far are we from there?

Proper spindle orientation is essential for cell fate determination and tissue morphogenesis. Recently, accumulating studies have elucidated several factors that regulate spindle orientation, including geometric, internal and external cues. Abnormality in these factors generally leads to defects in the physiological functions of various organs and the development of severe diseases. Herein, we first review models that are commonly used for studying spindle orientation. We then review a conservative heterotrimeric complex critically involved in spindle orientation regulation in different models. Finally, we summarize some cues that affect spindle orientation and explore whether we can establish a model that precisely elucidates the effects of spindle orientation without interfusing other spindle functions. We aim to summarize current models used in spindle orientation studies and discuss whether we can build a model that disturbs spindle orientation alone. This can substantially improve our understanding of how spindle orientation is regulated and provide insights to investigate this complex event.     

Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication.

Aneuploidy and polyploidy are commonly observed in transformed cells. These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome. The centrosome is the organizing center for the mitotic spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the spindle pole body. We review how defects in spindle pole body duplication or function lead to genetic instability in yeast. There are several well documented instances of genetic instability in yeast that can be traced to the spindle pole body, all of which serve as models for genetic instability in transformed cells.     

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gα_i1‐3 anchor the coiled‐coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gα_i1‐3 are dispensable for NuMA‐dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP₂ (PIP₂) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP₂ prevents NuMA cortical localization during mitosis, and conversely, increasing PIP₂ levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.     

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran—GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.     

Differences in Intrinsic Tubulin Dynamic Properties Contribute to Spindle Length Control in Xenopus Species

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A single internal telomere tract ensures meiotic spindle formation

Contact between telomeres and the fission yeast spindle pole body during meiotic prophase is crucial for subsequent spindle assembly, but the feature of telomeres that confers their ability to promote spindle formation remains mysterious. Here we show that while strains harbouring circular chromosomes devoid of telomere repeat tracts undergo aberrant meiosis with defective spindles, the insertion of a single internal telomere repeat stretch rescues the spindle defects. Moreover, the telomeric overhang‐binding protein Pot1 is dispensable for rescue of spindle formation. Hence, an inherent feature of the double‐strand telomeric region endows telomeres with the capacity to promote spindle formation.     

Attaching to spindles before they form: Do early incorrect chromosome-microtubule attachments promote meiotic segregation fidelity?

The proper partitioning of the genome during meiosis depends on the correct segregation of chromosomes. Errors in this process result in the production of aneuploid gametes, a major cause of birth defects and infertility in humans. In order to segregate properly in meiosis, homologous chromosome partners must attach to microtubules that emanate from opposites poles of the spindle. However, a recent study in yeast has shown that, remarkably, the initial attachments between microtubules and the chromosomes are usually incorrect, which would lead to catastrophic segregation errors, but they are nearly always corrected through the detachment and reattachment of the microtubules. Here we review the reasons for the initial incorrect attachments, which stem from the timing of their formation early in the spindle assembly process, and the fact that the microtubule organizers, called spindle pole bodies in yeast, are not equal. One spindle pole body is older and better able to produce microtubules that attach to the chromosomes. We draw parallels to recent findings in animal cells and suggest that these early microtubule attachments, while often incorrect, may serve an important role in spindle assembly, which, in the long-term, promotes high-fidelity chromosome segregation.     

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

Microtubule-interfering agents,spindle defects,and interkinetochore tension

Microtubule-interfering agents have been very useful both as biological tools in studying mitosis and as chemotherapeutic agents against cancer. It remains poorly understood how these agents converge on the spindle assembly checkpoint (SAC) to halt mitotic progression, while inhibiting microtubule dynamics by different mechanisms. Cells arrested at mitosis by various microtubule-interfering agents exhibit strikingly different defects in the mitotic spindle. However, all the arrested cells possess the 3F3/2 phosphoepitope at the sister kinetochores of chromosomes, indicating the decrease of tension across the paired kinetochores. In addition, microtubule-interfering agents result in a comparable reduction in the distance between sister kinetochores, suggesting that these agents decrease interkinetochore tension to similar degrees. Here, we discuss recent progress that suggests impairment of kinetochore-microtubule attachment and reduction of interkinetochore tension as common mechanisms underlying the persistent SAC activation in response to diverse microtubule-interfering agents.     

Parkin deficiency contributes to pancreatic tumorigenesis by inducing spindle multipolarity and misorientation

Parkin, an E3 ubiquitin ligase well known for its role in the pathogenesis of juvenile Parkinson disease, has been considered as a candidate tumor suppressor in certain types of cancer. It remains unknown whether parkin is involved in the development of pancreatic cancer, the fourth leading cause of cancer-related deaths worldwide. Herein, we demonstrate the downregulation and copy number loss of the parkin gene in human pancreatic cancer specimens. The expression of parkin negatively correlates with clinicopathological parameters indicating the malignancy of pancreatic cancer. In addition, knockdown of parkin expression promotes the proliferation and tumorigenic properties of pancreatic cancer cells both in vitro and in mice. We further find that parkin deficiency increases the proportion of cells with spindle multipolarity and multinucleation. Parkin-depleted cells also show a significant increase in spindle misorientation. These findings indicate crucial involvement of parkin deficiency in the pathogenesis of pancreatic cancer.     

单极纺锤体蛋白激酶1一个可能的新型抗肿瘤靶标

纺锤体装配检验点是有丝分裂分裂过程中一个非常重要的监督机器,其作用在于有丝分裂中期向后期转化前将所有的染色体排列到中期板上.近年来的研究表明,该检验点缺陷与肿瘤发生密切相关.单极纺锤体蛋白激酶1是纺锤体装配检验点的必需基因,存在于正常分裂细胞,并在肿瘤组织中高表达.最近研究发现,单极纺锤体蛋白激酶1的表达水平与乳腺癌恶性程度相关. 更有意思的是抑制其激酶活性或降低其蛋白水平将会导致多种肿瘤细胞的纺锤体装配检验点功能缺陷和细胞死亡.这表明,单极纺锤体蛋白激酶1是一个潜在的抗癌药物新靶标.本文对单极纺锤体蛋白激酶1如何调控纺锤体装配检验点以及其在抗肿瘤应用研究中的最新进展进行了回顾.     

Radial spindle and the phenotype of the maize meiotic mutant, dv

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.     

Successive Kinesin-5 Microtubule Crosslinking and Sliding Promote Fast,Irreversible Formation of a Stereotyped Bipolar Spindle

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Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

Microtubule organizing centers regulate spindle positioning in mouse oocytes

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