Microtubule motors in spindle and chromosome motility.

Many of the kinesin microtubule motor proteins discovered during the past 8-9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.     

CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.     

Microtubule motors in eukaryotic spindle assembly and maintenance

The spindle is a microtubule-based structure that facilitates chromosome segregation during mitosis and meiosis. Spindle assembly from dynamic microtubule building blocks is a major challenge for the dividing cell and a process that critically requires microtubule motors. In this review we focus on the mechanisms by which microtubule motors shape the spindle. Specifically, we address how motors are thought to move and arrange microtubules to form the characteristic bipolar morphology shared by all eukaryotic spindles as well as motor-dependent mechanisms of microtubule length regulation.     

Still entangled: Assembly of the central spindle by multiple microtubule modulators

The central spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the central spindle is formed, the role of several key proteins in this process and how central spindle assembly is temporally and spatially coordinated with mitosis.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

Separation of meiotic and mitotic effects of claret non-disjunctional on chromosome segregation in Drosophila.

The claret (ca) locus in Drosophila encodes a kinesin-related motor molecule that is required for proper distribution of chromosomes in meiosis in females and in the early mitotic divisions of the embryo. Here we demonstrate that a mutant allele of claret non-disjunctional (ca(nd)), non-claret disjunctional Dominant (ncdD), causes abnormalities in meiotic chromosome segregation, but is near wild-type with respect to early mitotic chromosome segregation. DNA sequence analysis of this mutant allele reveals two missense mutations compared with the predicted wild-type protein. One mutation lies in a proposed microtubule binding region of the motor domain and affects an amino acid residue that is conserved in all kinesin-related proteins reported to date. This region of the motor domain can be used to distinguish meiotic and mitotic motor function, defining an amino acid sequence criterion for classifying motors according to function. ncdD's mutant meiotic effect, but near wild-type mitotic effect, suggests that interactions of the ca motor protein with spindle microtubules differ in meiosis and mitosis.     

Structural and regulatory roles of nonmotor spindle proteins

Chromosome alignment and segregation during cell division rely on a highly ordered bipolar microtubule array called the mitotic spindle. The organization of microtubules into bipolar spindles with focused poles during mitosis requires numerous microtubule-associated proteins including both motor and nonmotor proteins. Nonmotor microtubule-associated proteins display extraordinary diversity in how they contribute to mitotic spindle organization. These mechanisms include regulation of microtubule nucleation and organization, direct and indirect influences on motor function, and control of cell cycle progression. Furthermore, many nonmotor spindle proteins display altered expression in cancer cells emphasizing their important roles in cell proliferation.     

Functional Roles of Poleward Microtubule Flux During Mitosis

Poleward microtubule flux is a conserved process during mitosis and meiosis in metazoan cells and is defined as the translocation of spindle microtubules toward spindle poles coupled to the depolymerization of their minus-ends. In some cell types, the rate of poleward microtubule flux matches that of poleward chromatid movement during anaphase A, suggesting that it pulls chromatids poleward. However, in other cell types, the rate of poleward microtubule flux is significantly slower than chromatid movement during anaphase A, suggesting that it makes little contribution to chromatid movement. This discrepancy led to speculation that flux is maintained in these cells to fulfill other functional roles aside from contributing to anaphase A chromatid movement. These roles include contributing to chromosome alignment, regulating spindle size and microtubule turnover, and correcting errors in chromosome attachment to spindle microtubules. Here, we discuss recent data that begin to pinpoint the functional roles of poleward microtubule flux during mitosis and meiosis.     

Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles

Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized to the spindle in early mitosis but to the pole-proximal ends of the spindle in anaphase B. The Klp5-motor and motor-neck localized to microtubules, often causing them to bundle. Over-expression of Klp6-motor or motor-neck resulted in shorter microtubules. These localization patterns were no different when constructs were expressed in strains lacking either or both of the endogenous, full-length proteins. Our results indicate that the localization signals for these kinesins are not derived from simple amino acid sequences but from complex interactions among multiple domains of each motor.     

Mechanisms of kinesin‐7 CENP‐E in kinetochore–microtubule capture and chromosome alignment during cell division

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere‐associated protein E (CENP‐E), a plus‐end‐directed kinesin motor, is required for congression of pole‐proximal chromosomes in metaphase. CENP‐E accumulates at the outer plate of kinetochores and mediates the kinetochore‐microtubule capture. CENP‐E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP‐E interacts with Bub1‐related kinase, Aurora B and core kinetochore components during kinetochore–microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin‐7 CENP‐E. We highlight the complicated interactions between CENP‐E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP‐E in mitosis and meiosis, including the kinetochore–microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP‐E in tumourigenesis and CENP‐E's specific inhibitors.     

Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.     

Roles and regulation of the Drosophila centromere cohesion protein MEI-S332 family

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of 相似文献   

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not in microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.     

MAPK-Activated Protein Kinase 2 Is Required for Mouse Meiotic Spindle Assembly and Kinetochore-Microtubule Attachment

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore–microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore–microtubule attachments.     

Mitotic spindle form and function

The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.     

Chromosome motion in mitosis.

The nature of the forces that move chromosomes in mitosis is beginning to be revealed. The kinetochore, a specialized structure situated at the primary constriction of the chromosome, appears to translocate in both directions along the microtubules of the mitotic spindle. One or more members of the newly described families of microtubule motor molecules may power these movements. Microtubules of the mitotic spindle undergo rapid cycles of assembly and disassembly. These microtubule dynamics may contribute toward generating force and regulating direction in chromosome movement.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

Accessory protein regulation of microtubule dynamics throughout the cell cycle

A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle.