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At least 19 recordsLinked to original sources
PubMed1982

Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP.

Permeabilized PtK1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs. ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase does no require exogenous ATP and is unaffected by these inhibitors. These results are consistent with "dynein-like" ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.

Adenosine Triphosphate↗
PubMed1977

[Partial disorganization of the anaphasic segregation of chromosomes in plant cells: combined actions of griseofulvin, producer of pluripolar anaphases and 2 ipecac alkaloids, producers of floating pole anaphases].

Anaphasis may be slightly checked by various treatments which however result in a normal chromosomic separation. Griseofulvin exerts a direct though partial influence on the mitotic apparatus, which entails "pluripolar anaphasis"; on the other hand Ipecac alkaloïds act indirectly and produce "floating poles anaphases". Treatments combining griseofulvin with cepheline or tubulosine show that there is never any synergy between the two processes. These results support our hypothesis that floating poles anaphases are not a sign of slight C-mitotic action but only come from a lag between the appearance/disappearance of microtubules and that of chromosomes during anaphasis.

Anaphase↗
PubMed1996

Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.

Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor. This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation. Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target. Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner. In addition, Pds1p is directly ubiquitinated by the Xenopus APC. In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase. We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase.

Anaphase↗
PubMed1994

Bypassing anaphase by fission yeast cut9 mutation: requirement of cut9+ to initiate anaphase.

A novel anaphase block phenotype was found in fission yeast temperature-sensitive cut9 mutants. Cells enter mitosis with chromosome condensation and short spindle formation, then block anaphase, but continue to progress into postanaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation, and cytokinesis. The cut9 mutants are defective in the onset of anaphase and possibly in the restraint of postanaphase events until the completion of anaphase. The cut9+ gene encodes a 78-kD protein containing the 10 34-amino acid repeats, tetratricopeptide repeats (TPR), and similar to budding yeast Cdc16. It is essential for viability, and the mutation sites reside in the TPR. The three genes, namely, nuc2+, scn1+, and scn2+, genetically interact with cut9+. The nuc2+ and cut9+ genes share an essential function to initiate anaphase. The cold-sensitive scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9.

Amino Acid Sequence↗
PubMed2001

Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p.

Kip1p of Saccharomyces cerevisiae is a bipolar kinesin in the conserved bimC kinesin subfamily that mediates mitotic spindle-pole separation. Here, we show that Kip1p is regulated immediately after anaphase initiation by its rapid degradation. Degradation required the ubiquitin protein ligase called the anaphase-promoting complex, the anaphase-promoting complex activating protein Cdc20, and a unique 43-aa sequence in Kip1p. Degradation also required import of Kip1p into the nucleus, but occurred independently of spindle association. A mutation that stabilized Kip1p impaired anaphase progression. The timing of degradation suggests that Kip1p functions primarily during spindle assembly and metaphase, and that Kip1p degradation facilitates structural changes in the mitotic spindle as anaphase progresses.

Amino Acid Sequence↗
PubMed2006

The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells.

Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

Anaphase↗
PubMed1994

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)-dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.

Anaphase↗
PubMed2005

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase.

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.

Anaphase↗
PubMed1983

Nocodazole pretreatment in anaphase selectively reduces anaphase B in PtK1 cells.

During early anaphase PtK1 cells were briefly treated with the rapidly reversible microtubule (MT) poison nocodazole. This treatment abruptly stopped chromosome motion and effected a large decrease in spindle birefringence. On removal of the drug, chromosome to pole motion (anaphase A) returned, though at a lesser rate but not extent than untreated cells. In most cases elongation of the pole-pole distance (anaphase B) also occurred, at both a rate and to an extent less than in untreated cells. During the recovery period following drug arrest spindle birefringence did not return to pretreatment levels. Electron microscopic analysis of nocodazole arrested, or arrested and released, cells revealed extensive disassembly of the nonkinetochore class of MTs (nkMTs), particularly evident in the astral region. Microtubules seen in the interzone region were largely fragments of midbody precursors. Kinetochore MTs (kMTs) appeared to be unaffected by the brief drug treatment chosen for these experiments. Analysis of MT profiles seen in transverse sections of the interzone region indicated in treated and released cells approximately 60% fewer MTs. This may suggest that chromosome motion during anaphase is not dependent on interactions between kMTs and nkMTs and separation of the spindle poles can occur in the presence of disrupted interzonal MTs.

Anaphase↗
PubMed1997

The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex.

The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis.

Amino Acid Sequence↗
PubMed1998

The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation.

The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1. We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC.

Anaphase↗
PubMed2026

The Small but Versatile Cyclin-Dependent Kinase Subunit CKS: C. elegans CKS-1 Regulates Anaphase Onset and Is Required for Anaphase B During Oocyte Meiosis.

The cyclin-dependent kinase subunit (CKS) remains the least well-understood component of the tripartite cyclin-dependent kinase (CDK) complexes that specify the proper sequence of events required to duplicate and segregate genomes during eukaryotic cell division. Our recent investigation of CKS requirements during Caenorhabditis elegans oocyte meiosis I and II documents an essential role for CKS in the execution of anaphase B and expands the known range of CKS influence on cell cycle regulation to a third class of E3 ligases. Our findings also further document how C. elegans oocytes use very different mechanisms to progress through these two sequential cell divisions that ultimately produce a haploid oocyte. In this Think Again article, we discuss our findings as they relate to previous studies of CKS in different model systems.

Animals↗
PubMed1988

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella.

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.6 micron/min, the rate of anaphase B is 2.3 +/- 0.3 micron/min, and the extent of anaphase B is 26.7 +/- 9.7% of metaphase spindle length. Colchicine was added to metaphase cells in order to inhibit any further addition of microtubule (MT) subunits onto the spindle. Colchicine, which does not break down the well-ordered Surirella central spindle, caused no significant change in the rate of anaphase A (1.3 +/- 0.3 micron/min) while it significantly decreased both the rate of anaphase B (1.2 +/- 0.4 micron/min) and the extent of anaphase B (14.8 +/- 8.3% of metaphase spindle length). Surirella cells were also treated with the metabolic inhibitor 2-4-dinitrophenol (DNP) in order to test the effects of energy depletion on anaphase. When DNP was added early in anaphase A, prior to the completion of sister chromosome separation, anaphase A was inhibited. When DNP was added after initiation of sister chromosome separation, anaphase A continued to completion, although at a lower rate than control cells (0.5 +/- 0.2 micron/min). Anaphase B was completely inhibited by DNP, but upon recovery from DNP resumed at a normal rate (2.2 +/- 0.5 micron/min) and progressed to a slightly larger than normal extent (44.0 +/- 13.0% of metaphase length).(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphase↗
PubMed2002

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism.

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.

Anaphase↗
PubMed1987

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.

Adenosine Triphosphate↗
PubMed1989

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase.

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.

Anaphase↗
PubMed1978

Mitosis in Barbulanympha. II. Dynamics of a two-stage anaphase, nuclear morphogenesis, and cytokinesis.

Anaphase in Barbulanympha proceeds in two discrete steps. In anaphase-A, chromosomal spindle fibers shorten and chromosomes move to the stationary centrosomes. In anaphase-B, the central spindle elongates and ("telophasic") bouquets of chromosomes, with kinetochores still connected by the shortened chromosomal fibers to the centrosomes, are moved far apart. The length, width, and birefringence of the central spindle remain unchanged throughout anaphase-A. In anaphase-B, the central spindle elongates up to fivefold. During elongation, the peripheral fibers of the central spindle splay, first anteriorly and then laterally. The remaining central spindle progressively becomes thinner and the retardation decreases; however, the coefficient of birefringence stays approximately constant. The nuclear envelope persists throughout mitosis in Barbulanympha and the nucleus undergoes an intricate morphological change. In prophase, the nucleus engulfs the spindle; in early anaphase-A, the nuclear envelope forms a seam anterior to the spindle, the nucleus thus transforms into a complete sleeve surrounding the central spindle. In late anaphase-A, the middle of the seam opens up in a cleft as the lips part; in anaphase-B, the cleft expands posteriorly, progressively exposing the central spindle. Finally, the cleft partitions the nucleus into two. The nuclear envelope shows an apparent elasticity and two-dimensional fluidity. Localized, transient deformations of the nuclear envelope indicate poleward and counter-poleward forces acting on the kinetochores embedded in the envelope. These forces appear responsible for nuclear morphogenesis as well as anaphase chromosome movement. At the end of anaphase-B, the two rostrate Barbulanympha may swim apart of be poked apart into two daughter cells by another organism cohabiting the host's hindgut.

Anaerobiosis↗