Showing papers on "Metaphase published in 2007"

Genes required for mitotic spindle assembly in Drosophila S2 cells.

Abstract: The formation of a metaphase spindle, a bipolar microtubule array with centrally aligned chromosomes, is a prerequisite for the faithful segregation of a cell's genetic material. Using a full-genome RNA interference screen of Drosophila S2 cells, we identified about 200 genes that contribute to spindle assembly, more than half of which were unexpected. The screen, in combination with a variety of secondary assays, led to new insights into how spindle microtubules are generated; how centrosomes are positioned; and how centrioles, centrosomes, and kinetochores are assembled.

Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore–microtubule interactions

Abstract: Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. Here, we demonstrate that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, we identify S676 as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, we show that this site is phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions.

Choice of Plk1 docking partners during mitosis and cytokinesis is controlled by the activation state of Cdk1

Abstract: Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.

Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

Abstract: The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod–Zw10–Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159–1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001–1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.

SIRT2, a tubulin deacetylase, acts to block the entry to chromosome condensation in response to mitotic stress.

Abstract: We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions.

Abstract: We previously demonstrated that a fraction of the human Nup107–160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107–160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107–160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107–160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore–microtubule attachment defects. Finally, we show that the presence of the Nup107–160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1–RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107–160 complex at kinetochores.

Cytoplasm-localized SIRT1 enhances apoptosis.

Abstract: In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.

Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.

Abstract: RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.

RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females.

Abstract: RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.

Changing Mad2 Levels Affects Chromosome Segregation and Spindle Assembly Checkpoint Control in Female Mouse Meiosis I

Abstract: The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/− oocytes, also leading to the generation of aneuploidies in meiosis I.

How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor

Abstract: Oocytes from higher chordates, including man and nearly all mammals, arrest at metaphase of the second meiotic division before fertilization. This arrest is due to an activity that has been termed 'Cytostatic Factor'. Cytostatic Factor maintains arrest through preventing loss in Maturation-Promoting Factor (MPF; CDK1/cyclin B). Physiologically, Cytostatic Factor – induced metaphase arrest is only broken by a Ca2+ rise initiated by the fertilizing sperm and results in degradation of cyclin B, the regulatory subunit of MPF through the Anaphase-Promoting Complex/Cyclosome (APC/C). Arrest at metaphase II may therefore be viewed as being maintained by inhibition of the APC/C, and Cytostatic Factor as being one or more pathways, one of which inhibits the APC/C, consorting in the preservation of MPF activity. Many studies over several years have implicated the c-Mos/MEK/MAPK pathway in the metaphase arrest of the two most widely studied vertebrates, frog and mouse. Murine downstream components of this cascade are not known but in frog involve members of the spindle assembly checkpoint, which act to inhibit the APC/C. Interesting these downstream components appear not to be involved in the arrest of mouse eggs, suggesting a lack of conservation with respect to c-Mos targets. However, the recent discovery of Emi2 as an egg specific APC/C inhibitor whose degradation is Ca2+ dependent has greatly increased our understanding of MetII arrest. Emi2 is involved in both the establishment and maintenance of metaphase II arrest in frog and mouse suggesting a conservation of metaphase II arrest. Its identity as the physiologically relevant APC/C inhibitor involved in Cytostatic Factor arrest prompted us to re-evaluate the role of the c-Mos pathway in metaphase II arrest. This review presents a model of Cytostatic Factor arrest, which is primarily induced by Emi2 mediated APC/C inhibition but which also requires the c-Mos pathway to set MPF levels within physiological limits, not too high to induce an arrest that cannot be broken, or too low to induce parthenogenesis.

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Abstract: Vertebrate oocytes are arrested in meiosis II until fertilization. This is one of two papers that link two previously known regulators of this arrest: the kinase Rsk that is activated by the Mos–MAPK pathway, directly phosphorylates Xerp1/Emi2 and thereby promotes its ability to inhibit the anaphase promoting complex APC/C. Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF)1, which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins—regulatory proteins of meiosis and mitosis—for degradation2,3. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest4,5,6, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest7 through activation of p90 ribosomal S6 kinase (p90rsk)8,9, is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box10, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint.

Abstract: The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (≈200 A long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are ≈5 μM. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the β-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.

Mitotic regulation of the anaphase-promoting complex

Abstract: Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C), a large multiprotein E3 ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome. APC/C has two activating subunits, Cdc20 and Cdh1. The well-established view is that Cdc20 activates APC/C from the onset of mitosis through the metaphase-anaphase transition, and that Cdh1 does so from anaphase through G1. Recent work, however, indicates that Cdh1 also activates APC/C in early mitosis and that this APC/C pool targets the anaphase inhibitor securin. To prevent premature degradation of securin, the nuclear transport factors Nup98 and Rae1 associate with APC/C(Cdh1)-securin complexes. In late metaphase, when all kinetochores are attached to spindle microtubules and the spindle assembly checkpoint is satisfied, Nup98 and Rae1 are released from these complexes, thereby allowing for prompt ubiquitination of securin by APC/C(Cdh1). This, and other mechanisms by which the catalytic activity of APC/C is tightly regulated to ensure proper timing of degradation of each of its mitotic substrates, are highlighted.

Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos

Abstract: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.

Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1).

Abstract: Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1–Cul1–F-box/βTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1–GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1–GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APCCdh1.

Discrete States of a Protein Interaction Network Govern Interphase and Mitotic Microtubule Dynamics

Abstract: The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.

Mitotic cell death by chromosome fragmentation

Abstract: Cell death plays a key role for both cancer progression and treatment. In this report, we characterize chromosome fragmentation, a new type of cell death that takes place during metaphase where condensed chromosomes are progressively degraded. It occurs spontaneously without any treatment in instances such as inherited status of genomic instability, or it can be induced by treatment with chemotherapeutics. It is observed within cell lines, tumors, and lymphocytes of cancer patients. The process of chromosome fragmentation results in loss of viability, but is apparently nonapoptotic and further differs from cellular death defined by mitotic catastrophe. Chromosome fragmentation represents an efficient means of induced cell death and is a clinically relevant biomarker of mitotic cell death. Chromosome fragmentation serves as a method to eliminate genomically unstable cells. Paradoxically, this process could result in genome aberrations common in cancer. The characterization of chromosome fragmentation may also shine light on the mechanism of chromosomal pulverization.

The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization

Abstract: A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly.

Depletion of topoisomerase IIα leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads

Abstract: Topoisomerase II (topo II) is a major component of mitotic chromosomes, and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/molecular mapping of the catalytic activity of topo II to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. By using a human cell line with a conditional-lethal mutation in the gene encoding DNA topoisomerase IIalpha, we find that depletion of topo IIalpha, while leading to a disorganised metaphase plate, does not have any overt effect on general assembly of kinetochores. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to depletion of topo IIalpha might be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH-coated connections between segregating chromatids.

Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function.

Abstract: Farnesyl transferase inhibitors (FTI) exhibit anticancer activity as a single agent in preclinical studies and show promise in combination with other therapeutics in clinical trials. Previous studies show that FTIs arrest cancer cells in mitosis; however, the mechanism by which this occurs is unclear. Here, we observed that treatment of various cancer cell lines with the FTI lonafarnib caused mitotic chromosomal alignment defects, leaving cells in a pseudometaphase state, whereby both aligned chromosomes and chromosomes juxtaposed to the spindle poles (termed ‘‘lagging chromosomes’’) were observed in the same cell. To determine how this occurs, we investigated the functionality of two farnesylated mitotic proteins, CENPE and CENP-F, which mediate chromosomal capture and alignment. The data show that lonafarnib in proliferating cancer cells depletes CENP-E and CENP-F from metaphase but not prometaphase kinetochores. Loss of CENP-E and CENP-F metaphase localization triggered aberrant chromosomal maintenance, causing aligned chromosomes to be prematurely released from the spindle equator and become lagging chromosomes, resulting in a mitotic delay. Furthermore, lonafarnib treatment reduces sister kinetochore tension and activates the BubR1 spindle checkpoint, suggesting that farnesylation of CENP-E and CENP-F is critical for their functionality in maintaining kinetochoremicrotubule interactions. Importantly, apparently similar chromosomal alignment defects were observed in head and neck tumors samples from a phase I trial with lonafarnib, providing support that lonafarnib disrupts chromosomal maintenance in human cancers. Lastly, to examine how farnesylation could regulate CENP-E in mediating kinetochore-microtubule attachments, we examined possible docking motifs of a farnesyl group on the outer surface of the microtubule. This analysis revealed three hydrophobic patches on the tubulin dimer for insertion of a farnesyl group, alluding to the possibility of an association between a farnesyl group and the microtubule. [Mol Cancer Ther 2007;6(4):1317 – 28]

Planar Spindle Orientation and Asymmetric Cytokinesis in the Mouse Small Intestine

Abstract: A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10° of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.

Intracellular calcium in the fertilization and development of mammalian eggs

Abstract: 1. Mammalian eggs are arrested at metaphase of their second meiotic division when ovulated and remain arrested until fertilized. The sperm delivers into the egg phospholipase C (PLC) zeta, which triggers a series of Ca(2+) spikes lasting several hours. The Ca(2+) spikes provide the necessary and sufficient trigger for all the events of fertilization, including exit from metaphase II arrest and extrusion of cortical granules that block the entry of other sperm. 2. The oscillatory Ca(2+) signal switches on calmodulin-dependent protein kinase II (CaMKII), which phosphorylates the egg-specific protein Emi2, earmarking it for degradation. To remain metaphase II arrested, eggs must maintain high levels of maturation-promoting factor (MPF) activity, a heterodimer of CDK1 and cyclin B1. Emi2 prevents loss of MPF by blocking cyclin B1 degradation, a process that is achieved by inhibiting the activity of the anaphase-promoting complex/cyclosome. However, CaMKII is not the primary initiator in the extrusion of cortical granules. 3. Ca(2+) spiking is also observed in mitosis of one-cell embryos, probably because PLCzeta contains a nuclear localization signal and so is released into the cytoplasm following nuclear envelope breakdown. The function of these mitotic Ca(2+) spikes remains obscure, although they are not absolutely required for passage through mitosis. 4. Intriguingly, the pattern of Ca(2+) spikes observed at fertilization has an effect on both pre- and postimplantation development in a manner that is independent of their ability to activate eggs. This suggests that the Ca(2+) spikes set in train at fertilization are having effects on processes initiated in the newly fertilized egg but whose influences are only observed several cell divisions later. The nature of the signals remains little explored, but their importance is clear and so warrants further investigation.