Central Spindle Microtubules Research Articles

Dynamic Localization of the Human Papillomavirus Type 11 Origin Binding Protein E2 through Mitosis While in Association with the Spindle Apparatus

Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.

Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis

During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol we recently developed, we show that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. We also demonstrate that both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. We find that centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins.

The meiotic spindle of theDrosophilaoocyte: the role of Centrosomin and the central aster

We provide here the first evidence that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. We also identified the core component centrosomal protein centrosomin (CNN) at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. We discuss the proposal that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles.

Modulated targeting of GFP‐AtMAP65‐1 to central spindle microtubules during division

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.

Klp67A destabilises pre-anaphase microtubules but subsequently is required to stabilise the central spindle

Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted and regressed as they could not be sustained by the central spindles that became increasing unstable over time and often completely degraded. Consistent with this, actin and anillin failed to form homogenous bands. Thus, the Klp67A microtubule catastrophe factor is required for cytokinesis by regulating both the formation and stability of the central spindle.

Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy

Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other organisms.

Cytokinesis: The Central Spindle Takes Center Stage

The central spindle plays a key role in cytokinesis. Recent studies have shed new light on how assembly of the central spindle is regulated, and also support a role for both the central spindle and astral microtubules in cytokinesis in animal cells.

Characterization of mid-spindle microtubules during furrow positioning in early cleavage period zebrafish embryos

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle 'pre-furrowing microtubule array' that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

Inside and Outside the Beltway

CELL BIOLOGY Microtubules form the mitotic spindle, which is the scaffold on which chromosomes are separated. Inoue et al. wanted to understand what microtubules might do after the partitioning of the chromosomes is complete; in the final stage of cell division, cytokinesis, the two daughter cells are physically separated from one another. Looking at primary spermatocytes from Drosophila melanogaster with time-lapse microscopy, they find that at the onset of cytokinesis the central spindle has two populations of microtubules—some localized to the interior of the central spindle and others extending toward the periphery—which are affected differentially in the presence of a mutant form of the microtubule-associated protein Orbit (aka Mast or CLASP). Normally, Orbit localizes to interior rather than peripheral central spindle microtubules, but in hypomorphic orbit mutants these interior microtubules become less stable. The remaining peripheral microtubules can still probe the cell cortex, where they help to initiate the cleavage furrow. However, some of the other components involved in cytokinesis fail to assemble properly, and ultimately the furrow regresses, yielding multinucleate cells. — SMH J. Cell Biol. 166 , 49 (2004).

Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression.

We address the relative roles of astral and central spindle microtubules (MTs) in cytokinesis of Drosophila melanogaster primary spermatocytes. Time-lapse imaging studies reveal that the central spindle is comprised of two MT populations, “interior” central spindle MTs found within the spindle envelope and “peripheral” astral MTs that probe the cytoplasm and initiate cleavage furrows where they contact the cortex and form overlapping bundles. The MT-associated protein Orbit/Mast/CLASP concentrates on interior rather than peripheral central spindle MTs. Interior MTs are preferentially affected in hypomorphic orbit mutants, and consequently the interior central spindle fails to form or is unstable. In contrast, peripheral MTs still probe the cortex and form regions of overlap that recruit the Pav-KLP motor and Aurora B kinase. orbit mutants have disorganized or incomplete anillin and actin rings, and although cleavage furrows initiate, they ultimately regress. Our work identifies a new function for Orbit/Mast/CLASP and identifies a novel MT population involved in cleavage furrow initiation.

NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization.

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

Linking spindle to furrow

Once it gets going, cytokinesis is a simple squeeze. The complicated machinery is devoted instead to localizing the cytokinesis furrow. Now, Gregory Somers and Robert Saint (Australian National University, Canberra, Australia) have provided a link from mitotic spindle to contractile furrow that may explain how one positions the other. Figure Rings of RacGAP50C (red) and PBL (green) link spindle and furrow. The group started with the furrow-localized protein Pebble (PBL), a guanine nucleotide exchange factor (GEF) and thus activator for Rho1 and actin remodeling. Two-hybrid and coimmunoprecipitation experiments with PBL turned up RacGAP50C. The worm homologue of RacGAP50C, CYK-4, is essential for cytokinesis completion and binds a kinesin-like protein that bundles microtubules in the central spindle. Consistent with these interactions, RacGAP50C was found in an inner ring near central spindle microtubules, abutting an outer ring of PBL. How these rings affect each other is unclear. PBL and RacGAP50C, despite being an activating GEF and an inhibitory GTPase-activating protein (GAP), interact synergistically rather than antagonistically. Somers and Saint find that the GAP activity is not directed at Rho1 but is required for cytokinesis. Perhaps the logic behind the activity will be tied up in the need to regulate the timing of PBL activation. ▪ Reference: Somers, W.G., and R. Saint. 2003. Dev. Cell. 4:29–39. [PubMed]

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells.

We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis.

Meics, a novel zinc-finger protein which relocates from nuclei to the central meiotic spindle during Drosophila spermatogenesis.

A Drosophila gene encoding a novel zinc-finger protein, Meics, was cloned using a monoclonal antibody. The predicted amino acid sequence contains 12 zinc-finger motifs of the C2H2-type. During spermatogenesis, Meics distributes intranuclearly at pre- and post-meiotic stages whereas it relocates to central-spindle microtubules at both meiotic divisions.

The Drosophila Protein Asp Is Involved in Microtubule Organization during Spindle Formation and Cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells.

Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other.

A motor goes mitotic and divorces the Golgi complex

Use of two-hybrid screening has previously shown that the Rab6-binding kinesin Rab6-KIFL is a partner of Rab6, a Golgi-associated GTPase implicated in intra-Golgi membrane traffic. Hill et al.1xThe Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis. Hill, E. et al. EMBO J. 2000; 19: 5711–5719Crossref | PubMedSee all References1 have now characterized the expression, localization and tentative function of human Rab6-KIFL. Rab6-KIFL accumulates in mitotic HeLa cells, where it localizes to central spindle microtubules and to the midbody during cytokinesis. Using an antiserum raised against the Rab6-binding domain of Rab6-KIFL, Hill et al. demonstrate that levels of the protein oscillate in a cell-cycle-controlled manner, mirroring the behavior of cyclin B2. This suggests that Rab6-KIFL is a kinesin found solely in cells during mitosis. Most interestingly, microinjection of antibodies against Rab6-KIFL blocks cytokinesis and results in a dramatic accumulation of binucleate cells. Furthermore, overexpression of Rab6-KIFL in HeLa cells causes cell death. Thus, Rab6-KIFL might be a microtubule motor with functions during formation of the cleavage furrow and cytokinesis.These new data on Rab6-KIFL create a conundrum as we had grown accustomed to think about this protein more as a Golgi-associated motor protein involved in vesicular traffic in interphase cells 2xInteraction of a Golgi-associated kinesin-like protein with Rab6. Echard, A. et al. Science. 1998; 279: 580–585Crossref | PubMed | Scopus (349)See all References2. Echard et al.2xInteraction of a Golgi-associated kinesin-like protein with Rab6. Echard, A. et al. Science. 1998; 279: 580–585Crossref | PubMed | Scopus (349)See all References2 had demonstrated a staining of the Golgi complex in interphase HeLa cells with an antiserum raised against the same region of (mouse) Rab6-KIFL as used by Hill et al. Intriguingly, any such Golgi staining pattern is absent with the new Rab6-KIFL antiserum raised by Hill et al. There are several possible explanations for this difference. The antiserum used by Echard et al. might contain a cross-reactivity against a Golgi-associated protein that masks the real localization of Rab6-KIFL. Indeed, Hill et al. show that their antiserum recognizes only the 100-kDa band of Rab6-KIFL, whereas the antiserum raised by Echard et al. additionally sees a band of 200 kDa. However, a much more interesting possibility could perhaps be that the two laboratories have characterized highly related but functionally distinct isoforms of Rab6-KIFL that perform functions in mitotic and interphase cells, respectively. Future experiments will show us whether Rab6-KIFL has divorced the Golgi complex or whether we might yet see a reconciliation.

Relationships between the central spindle and the contractile ring during cytokinesis in animal cells

During late anaphase and telophase, animal cells develop a bundle of antiparallel, interdigitating microtubules between the two daughter nuclei. Recent data indicate that this structure, called the central spindle, plays an essential role during cytokinesis. Studies in Drosophila and on vertebrate cells strongly suggest that the molecular signals for cytokinesis specifically emanate from the central spindle midzone. Moreover, the analysis of Drosophila mutants defective in cytokinesis has revealed a cooperative interaction between the central spindle microtubules and the contractile ring: when either of these structures is perturbed, the proper assembly of the other is disrupted. Based on these results we propose a model for the role of the central spindle during cytokinesis. We suggest that the interaction between central spindle microtubules and cortical actin filaments leads to two early events crucial for cytokinesis: the positioning of the contractile ring, and the stabilization of the plus ends of the interdigitating microtubules that comprise the central spindle. The latter event would provide the cell with a specialized microtubule scaffold that could mediate the translocation of plus-end-directed molecular motors to the cell's equator. Among the cargoes transported by these motors could be proteins involved in the regulation and execution of cytokinesis.

Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis.

We analyzed male meiosis in mutants of the chickadee (chic) locus, a Drosophila melanogaster gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. During ana-telophase of both meiotic divisions, they exhibit a central spindle less dense than wild type; certain chic allelic combinations cause almost complete disappearance of the central spindle. Moreover, chic mutant spermatocytes fail to form an actomyosin contractile ring. To further investigate the relationships between the central spindle and the contractile ring, we examined meiosis in the cytokinesis-defective mutants KLP3A and diaphanous and in testes treated with cytochalasin B. In all cases, we found that the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: When one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, we observed another consequence of chic mutations: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus, a gene that encodes a Drosophila member of the cofilin/ADF family of actin-severing proteins. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration.

Kinesin-related KIP3 of Saccharomyces cerevisiae is required for a distinct step in nuclear migration.

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.