Anaphase Research Articles

Successive recruitment of p-CDC25B-Ser351 and p-cyclin B1-Ser123 to centrosomes contributes to the release of mouse oocytes from prophase I arrest.

The molecular mechanism that controls the activation of Cyclin B1-CDK1 complex has been widely investigated. It is generally believed that CDC25B acts as a "starter phosphatase" of mitosis. In this study, we investigate the sequential regulation of meiotic resumption by CDC25B and Cyclin B1 in mouse oocytes. Injection of mRNAs coding for CDC25B-Ser351A and/or Cyclin B1-Ser123A shows a more potent maturation-inhibiting ability than their respective wild type. Co-injection of mRNAs coding for phosphor-mimic CDC25B-Ser351D and Cyclin B1-Ser123D can rescue this prophase I arrest induced by CDC25B-Ser351A or Cyclin B1-Ser123A. In addition, p-CDC25B-Ser351 is co-localized at the microtubule-organizing centers (MTOCs) with Aurora kinase A (AURKA) during maturation and p-Cyclin B1-Ser123 is only captured on MTOCs shortly before germinal vesicle breakdown (GVBD). Depletion of AURKA not only resulted in metaphase I (MI) spindle defects and anaphase I (AI) abnormal chromosomes separation but also prevented the phosphorylation of CDC25B-Ser351 at centrosomes. AURKA depletion induced deficiencies of spindle assembly and progression to MII can be rescued by CDC25B-Ser351D mRNA injection. AURKA induced phosphorylation and recruitment of CDC25B to MTOCs prior to p-Cyclin B1-Ser123, and this sequential regulation is essential for the commitment of the oocytes to resume meiosis.

Regulation of mitotic spindle disassembly by an environmental stress-sensing pathway in budding yeast.

Timely spindle disassembly is essential for coordination of mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to microtubule depolymerization and shrinkage of spindle halves. By examining the genetic interactions of known spindle disassembly genes, we identified three genes in the environmental stress-sensing HOG (high-osmolarity glycerol response) pathway, SHO1, PBS2, and HOG1, and found they are necessary for proper localization of She1 to the anaphase spindle and for proper spindle disassembly. HOG pathway mutants exhibited spindle disassembly defects, as well as mislocalization of anillin-related proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places Boi2 in a pathway with Sho1, Pbs2, and Hog1. Together, our data identify a process by which cells monitor events at the spindle and bud neck and describe a novel role for the HOG pathway in mitotic signaling.

The long and viscous road: uncovering nuclear diffusion barriers in closed mitosis.

Diffusion barriers are effective means for constraining protein lateral exchange in cellular membranes. In Saccharomyces cerevisiae, they have been shown to sustain parental identity through asymmetric segregation of ageing factors during closed mitosis. Even though barriers have been extensively studied in the plasma membrane, their identity and organization within the nucleus remains poorly understood. Based on different lines of experimental evidence, we present a model of the composition and structural organization of a nuclear diffusion barrier during anaphase. By means of spatial stochastic simulations, we propose how specialised lipid domains, protein rings, and morphological changes of the nucleus may coordinate to restrict protein exchange between mother and daughter nuclear lobes. We explore distinct, plausible configurations of these diffusion barriers and offer testable predictions regarding their protein exclusion properties and the diffusion regimes they generate. Our model predicts that, while a specialised lipid domain and an immobile protein ring at the bud neck can compartmentalize the nucleus during early anaphase; a specialised lipid domain spanning the elongated bridge between lobes would be entirely sufficient during late anaphase. Our work shows how complex nuclear diffusion barriers in closed mitosis may arise from simple nanoscale biophysical interactions.

Budding yeast kinetochore proteins, Chl4 and Ctf19, are required to maintain SPB-centromere proximity during G1 and late anaphase.

In the budding yeast, centromeres stay clustered near the spindle pole bodies (SPBs) through most of the cell cycle. This SPB-centromere proximity requires microtubules and functional kinetochores, which are protein complexes formed on the centromeres and capable of binding microtubules. The clustering is suggested by earlier studies to depend also on protein-protein interactions between SPB and kinetochore components. Previously it has been shown that the absence of non-essential kinetochore proteins of the Ctf19 complex weakens kinetochore-microtubule interaction, but whether this compromised interaction affects centromere/kinetochore positioning inside the nucleus is unknown. We found that in G1 and in late anaphase, SPB-centromere proximity was disturbed in mutant cells lacking Ctf19 complex members,Chl4p and/or Ctf19p, whose centromeres lay further away from their SPBs than those of the wild-type cells. We unequivocally show that the SPB-centromere proximity and distances are not dependent on physical interactions between SPB and kinetochore components, but involve microtubule-dependent forces only. Further insight on the positional difference between wild-type and mutant kinetochores was gained by generating computational models governed by (1) independently regulated, but constant kinetochore microtubule (kMT) dynamics, (2) poleward tension on kinetochore and the antagonistic polar ejection force and (3) length and force dependent kMT dynamics. Numerical data obtained from the third model concurs with experimental results and suggests that the absence of Chl4p and/or Ctf19p increases the penetration depth of a growing kMT inside the kinetochore and increases the rescue frequency of a depolymerizing kMT. Both the processes result in increased distance between SPB and centromere.

Different Patterns of Chromosomal Histone H3 Phosphorylation in Land Plants

The chromosomal phosphorylation of histone H3 during mitosis and meiosis seems to play a fundamental role in the control of cell division in all eukaryotes. In plants, the temporal and spatial distribution of H3S10 phosphorylated (H3S10ph) is currently known only for chromosomes of a few angiosperms. In the present study, we analyzed the pattern of H3S10ph in mitotic chromosomes of 14 plant species, including 2 bryophytes and 12 tracheophytes. In all species, the phosphorylation of H3S10 was cytologically detected first in prophase and disappeared in late anaphase. Two patterns of chromosomal phosphorylation were observed: (1) only the pericentromeric regions were hyperphosphorylated, whereas the remaining chromosome arms appeared weakly and diffusely immunolabeled, and (2) the whole chromosomes appear uniformly phosphorylated, increasing the labeling intensity at metaphase. The first pattern was observed in all tracheophytes with monocentric chromosomes, whereas the second one was restricted to the bryophytes and angiosperms with holokinetic chromosomes. In both cases, no particular kind of chromatin such as NORs or heterochromatic bands were differentially labeled. Based on this data and previous analyses in other eukaryotes, we suggest that hyperphosphorylation of the whole mitotic metaphase chromosome represents the ancestral condition for eukaryotic chromosomes, and the change to the pericentromeric pattern occurred in the transition from bryophytes to tracheophytes. The meaning and possible implications of these results are discussed in the light of recent and classical experiments.

Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called “anaphase problem” is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore–microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear. Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/CCdc20 substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/CCdc20 activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10–15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/CCdc20 in late anaphase helps to keep cyclin B1 levels low.

The budding yeast amphiphysin complex is required for contractile actin ring (CAR) assembly and post-contraction GEF-independent accumulation of Rho1-GTP.

The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR), primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect.

Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis.

Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

PP 2A delays APC /C‐dependent degradation of separase‐associated but not free securin

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2- or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpoint-mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.

Cytokinesis requires supervillin‐mediated binding to myosin II and synergism with anillin (962.1)

Cytokinesis requires coordination of cortical myosin II activation with central spindle alignment. Supervillin and anillin are two actin‐ and myosin II‐associated vertebrate proteins implicated in maintenance of the cytokinetic furrow in early cytokinesis, The nature of their cross‐talk is unknown. Point mutations within an evolutionarily conserved sequence in supervillin, residues 99 ‐ 153, eliminate binding to the nonmuscle myosin II heavy chain (MHC), confer dominant‐negative inhibition of cytokinesis, and abrogate the ability of full‐length supervillin to rescue RNAi‐mediated depletion of supervillin. While an observed ~2‐fold increase of anillin in supervillin‐knockdown cells does not eliminate cytokinetic failure, knockdown of both proteins together yields more than additive increases in numbers of multinucleated cells, as compared with depletion of either protein alone. Localizations of supervillin, anillin, MHC (total myosin), and phosphorylated regulatory myosin II light chain (pMRLC, activated myosin) in dividing cells suggest that supervillin and anillin synergize to confine MHC to the furrow. Anillin alone is sufficient to maintain normal levels of pMRLC at the furrow in late anaphase, but supervillin is required for normal pMRLC localization in early anaphase and around the cytokinetic bridge during daughter cell separation. Protein affinity isolations of GFP‐tagged supervillin and anillin indicate that these proteins do not significantly associate with each other although myosin II, actin and other cytoskeletal proteins co‐purify with each bait protein. None of these proteins co‐isolated with GFP alone. Stark differences between specifically co‐isolating kinases, phosphatases, and other signaling proteins reinforce the conclusion that supervillin and anillin regulate cytokinesis through separate pathways.Grant Funding Source: Supported by the National Institutes of Health and the University of Massachusetts Medical School.

Cdc14 targets the Holliday junction resolvase Yen1 to the nucleus in early anaphase

The only canonical Holliday junction (HJ) resolvase identified in eukaryotes thus far is Yen1/GEN1. Nevertheless, Yen1/GEN1 appears to have a minor role in HJ resolution, and, instead, other structure-specific endonucleases (SSE) that recognize branched DNA play the leading roles, Mus81-Mms4/EME1 being the most important in budding yeast. Interestingly, cells tightly regulate the activity of each HJ resolvase during the yeast cell cycle. Thus, Mus81-Mms4 is activated in G2/M, while Yen1 gets activated shortly afterwards. Nevertheless, cytological studies have shown that Yen1 is sequestered out of the nucleus when cyclin-dependent kinase activity is high, i.e., all of the cell cycle but G1. We here show that the mitotic master phosphatase Cdc14 targets Yen1 to the nucleus in early anaphase through the FEAR network. We will further show that this FEAR-mediated Cdc14-driven event is sufficient to back-up Mus81-Mms4 in removing branched DNA structures, which are especially found in the long chromosome arms upon replication stress. Finally, we found that MEN-driven Cdc14 re-activation in late anaphase is essential to keep Yen1 in the nucleus until the next G1. Our results highlight the essential role that early-activated Cdc14, i.e., through the FEAR network, has in removing all kind of non-proteinaceous linkages that preclude faithful sister chromatid segregation in anaphase. In addition, our results support the general idea of Yen1 acting as a last resource endonuclease to deal with any remaining HJ that might compromise genetic stability during chromosome segregation.

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.

The Mitotic Exit Network: new turns on old pathways

In budding yeast, the Mitotic Exit Network (MEN) is a signaling pathway known to drive cells out of mitosis and promote the faithful division of cells. The MEN triggers inactivation of cyclin-dependent kinase (Cdk1), the master regulator of mitosis, and the onset of cytokinesis after segregation of the daughter nuclei. The current model of the MEN suggests that MEN activity is restricted to late anaphase and coordinated with proper alignment of the spindle pole bodies (SPBs) with the division axis. However, recent evidence suggests that MEN activity may function earlier in mitosis, prompting re-evaluation of the current model. Here we attempt to integrate this recent progress into the current view of mitotic exit.

The FEAR Protein Slk19 Restricts Cdc14 Phosphatase to the Nucleus until the End of Anaphase, Regulating Its Participation in Mitotic Exit in Saccharomyces cerevisiae

In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk193R (three lysine-to-arginine mutations), strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk193R cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk193R cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk193R cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk193R causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk193R partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.

Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II

Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.

Lre1 Directly Inhibits the NDR/Lats Kinase Cbk1 at the Cell Division Site in a Phosphorylation-Dependent Manner

The nuclear Dbf2 related (NDR) family of protein kinases play important roles in cell-cycle regulation, apoptosis, cell morphogenesis, and development in a variety of organisms. In budding yeast, the NDR kinase complex composed of Cbk1 and its regulatory subunit, Mob2, have an established role in the control of cell separation/abscission that follows cytokinesis. Whereas the activators of Cbk1-Mob2 have been more extensively described, the mechanisms that restrict or inhibit Cbk1-Mob2 catalytic activity remain largely unknown. We identified the protein Lre1 as a direct inhibitor of Cbk1-Mob2 catalytic activity. We show that Lre1 accumulates at the cell division site in late anaphase and associates with both Mob2 and Cbk1 in vivo and in vitro. Biochemical and functional analysis established that the ability of Lre1 to associate with Cbk1-Mob2 was reduced by mitotic Cdk1 activity and promoted by Cdc14 phosphatase at the end of mitosis. The inhibition of Cbk1-Mob2 by Lre1 was critical to promote the survival of cells lacking the actomyosin driven pathway of cytokinesis. We established Lre1 as a direct inhibitor of the NDR kinase Cbk1-Mob2, which is regulated in a cell-cycle-dependent manner. We propose that similar inhibitory proteins may also provide fine tuning for the activity of NDR kinases in other organisms.

Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

The Budding Yeast Point Centromere Associates with Two Cse4 Molecules during Mitosis

The centromere is defined by the incorporation of the centromere-specific histone H3 variant centromere protein A (CENP-A). Like histone H3, CENP-A can form CENP-A-H4 heterotetramers in vitro. However, the in vivo conformation of CENP-A chromatin has been proposed by different studies as hemisomes, canonical, or heterotypic nucleosomes. A clear understanding of the in vivo architecture of CENP-A chromatin is important, because it influences the molecular mechanisms of the assembly and maintenance of the centromere and its function in kinetochore nucleation. A key determinant of this architecture is the number of CENP-A molecules bound to the centromere. Accurate measurement of this number can limit possible centromere architectures. The genetically defined point centromere in the budding yeast Saccharomyces cerevisiae provides a unique opportunity to define this number accurately, as this 120-bp-long centromere can at the most form one nucleosome or hemisome. Using novel live-cell fluorescence microscopy assays, we demonstrate that the budding yeast centromere recruits two Cse4 (ScCENP-A) molecules. These molecules are deposited during S phase and they remain stably bound through late anaphase. Our studies suggest that the budding yeast centromere incorporates a Cse4-H4 tetramer.

The maternal‐to‐zygotic transition: An asynchronous split

AbstractThe early cell cycles of preimplantation embryo development are unique in the scheme of mitotic cell proliferation as cell division is not coupled to cell growth, leading to a halving of blastomere volume with each cleavage event. Among the early mouse embryonic divisions, the fi rst two are particularly different, lasting almost twice as long as subsequent divisions. The third cell cycle is marked by the transition of a four‐cell embryo into an eight‐cell embryo, and represents the fi rst complete cell cycle occurring after activation of the zygotic genome. The G2/M phase of the third cell cycle is highly variable, lasting between 2–5 hours, and heterogeneity between blastomeres within the same embryo may occur as a part of normal development.The embryo in this image is actively undergoing cleavage from the four‐ to the eight‐cell stage, and blastomeres are captured in multiple phases of the cell cycle, as visualized by chromatin structure (DNA, blue) and microtubule staining (α‐tubulin, green). Two blastomeres sit in interphase with decondensed chromatin masses and a mesh‐like microtubule network, while the remaining blastomeres are actively undergoing mitosis. Of the latter, one is in metaphase, one in early anaphase, and the last in late anaphase. All together, the diversity in cell cycle stages reveals the inherit asynchrony existent within individual blastomeres of a cleavage stage embryo. Mol. Reprod. Dev. 80: 1–1, 2012. © 2012 Wiley Periodicals, Inc.

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

In this study, we examined the anaphase functions of the S. cerevisiae kinesin-5 homolog Kip1. We show that Kip1 is attached to the mitotic spindle midzone during late anaphase. This attachment is essential to stabilize interpolar microtubule (iMTs) plus-ends. By detailed examination of iMT dynamics we show that at the end of anaphase, iMTs depolymerize in two stages: during the first stage, one pair of anti-parallel iMTs depolymerizes at a velocity of 7.7 µm/minute; during the second stage, ∼90 seconds later, the remaining pair of iMTs depolymerizes at a slower velocity of 5.4 µm/minute. We show that upon the second depolymerization stage, which coincides with spindle breakdown, Kip1 follows the plus-ends of depolymerizing iMTs and translocates toward the spindle poles. This movement is independent of mitotic microtubule motor proteins or the major plus-end binding or tracking proteins. In addition, we show that Kip1 processively tracks the plus-ends of growing and shrinking MTs, both inside and outside the nucleus. The plus-end tracking activity of Kip1 requires its catalytic motor function, because a rigor mutant of Kip1 does not exhibit this activity. Finally, we show that Kip1 is a bi-directional motor: in vitro, at high ionic strength conditions, single Kip1 molecules move processively in the minus-end direction of the MTs, whereas in a multi-motor gliding assay, Kip1 is plus-end directed. The bi-directionality and plus-end tracking activity of Kip1, properties revealed here for the first time, allow Kip1 to perform its multiple functions in mitotic spindle dynamics and to partition the 2-micron plasmid.