Correspondence to: Don W. Cleveland, Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla, CA 92093. Tel:(858) 534-7811 Fax:(858) 534-7659 E-mail:dcleveland{at}ucsd.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CENP-meta has been identified as an essential, kinesin-like motor protein in Drosophila. The 257-kD CENP-meta protein is most similar to the vertebrate kinetochore-associated kinesin-like protein CENP-E, and like CENP-E, is shown to be a component of centromeric/kinetochore regions of Drosophila chromosomes. However, unlike CENP-E, which leaves the centromere/kinetochore region at the end of anaphase A, the CENP-meta protein remains associated with the centromeric/kinetochore region of the chromosome during all stages of the Drosophila cell cycle. P-elementmediated disruption of the CENP-meta gene leads to late larval/pupal stage lethality with incomplete chromosome alignment at metaphase. Complete removal of CENP-meta from the female germline leads to lethality in early embryos resulting from defects in metaphase chromosome alignment. Real-time imaging of these mutants with GFP-labeled chromosomes demonstrates that CENP-meta is required for the maintenance of chromosomes at the metaphase plate, demonstrating that the functions required to establish and maintain chromosome congression have distinguishable requirements.
Key Words: kinetochore, kinesin-like protein, CENP-E, chromosome congression, spindle assembly checkpoint
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Precise chromosome partitioning during mitosis requires a specialized microtubule-based structure, the mitotic spindle. The spindle interacts with chromosomes most strongly via a protein complex called the kinetochore, which forms at the centromere of each chromosome during cell division (see
CENP-E was first identified as a protein that is present on the kinetochores of mitotic cells during chromosome movement (
The presence of this kinesin-like motor at the kinetochore during mitosis makes CENP-E an obvious candidate for providing motive force for any of a number of different chromosome movements. Consistent with this view, microinjection of cells with antibodies specific for CENP-E caused a delay in anaphase onset (
Additional observations suggest the possibility that CENP-E may have other roles during mitosis. For example, the relocalization of CENP-E to the midzone at anaphase-B may indicate an additional role for this plus-end motor (
To examine the role of kinetochore-associated kinesin motors in mitosis in vivo, especially in distinguishing whether kinetochore kinesins like CENP-E are required for the establishment of chromosome congression or its maintenance once chromosomes are properly aligned, we have now used genetic methods to determine the in vivo consequences of removal of one of two kinesin relatives of CENP-E in Drosophila.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of CENP-meta and CENP-ana cDNAs and Genomic DNA Constructs
A small region corresponding to amino acids (aa)1 81130 of the amino terminus of both CENP-meta and CENP-ana was identified in a PCR-based screen for new members of the kinesin superfamily in Drosophila. A 198-bp oligonucleotide probe (58 nucleotides [nts] of intron 2 followed by codons for aa 87130 of both proteins) was synthesized corresponding to this sequence and used to probe a Drosophila genomic P1 library filter (Genome Systems, Inc.). All DNA hybridizations were performed at 60°C according to
|
cDNA clones for CENP-meta and CENP-ana were isolated from a Drosophila embryonic ZAP library (Stratagene) and a Drosophila 04 h embryonic library (a kind gift from Nick Brown;
Complete predicted coding sequences for CENP-meta and CENP-ana are available from GenBank/EMBL/DDBJ under accession numbers AF220353 and AF220354, respectively.
Mutants and Derivatives
P-element lines from the Bloomington stock center and Tod Laverty at BDGP were screened for insertions at 32E in the CENP-meta gene. Genomic sequences around the P-element were isolated by plasmid rescue. Plasmid rescue was used to identify P-element l(2)04431 within the CENP-meta region. Subsequent sequencing of the rescued sequences and of inverse PCR (invPCR) products (BDGP protocol) containing the same flanking sequences allowed precise assignment of the insertion point of l(2)04431 within exon 10 of CENP-meta gene. Excision, transposition, and deletion lines were obtained by crossing the l(2)04431 strain into a background containing the 2-3 transposase source. Transposition of l(2)04431 in line CENP-meta
deleted
5 kb of genomic sequence in the CENP-meta gene just upstream of the 5' end of the initial P-element insertion site.
To generate germ-line clones of CENP-meta, cmet-FRT chromosomes were constructed by recombination of P[ry+;neoR-FRT] 40A; ry with cmet/SM1; ry lines. Germ-line clones were induced using FRT-ovoD1 and hsFLPase following
RNA Blot Analysis
Total RNA was isolated from embryos and female adults using Trizol reagent (GIBCO BRL). 15 µg of total RNA from adult females and 30 µg of total RNA from embryos from a variety of strains were run on a 1.0% denaturing agarose/formaldehyde gel as described in Current Protocols in Molecular Biology. The RNA was transferred to Hybond (Amersham Pharmacia Biotech) and hybridized in Church and Gilbert buffer at 60°C, overnight. Initially the filter was hybridized with a 686-bp BglII-BamHI fragment (nt 6,2486,934) specific for CENP-meta. After exposure to film the filter was stripped until there was no detectable signal. The filter was then hybridized with a 3-kb probe to the Drosophila mcm5 gene (ZAP 4-2, which recognizes CENP-ana (and weakly cross-reacts with CENP-meta), then stripped and probed for PPT-2 with a genomic PCR fragment corresponding to the first four exons of PPT-2. All blots were exposed to XAR-XOMAT film (Kodak) at -80°C with intensifying screens.
Expression and Purification of CENP-meta in E. coli
The vector pQE 30 (QIAGEN) was used to express an NH2-terminal hexahistidine fusion with aa 285706 of CENP-meta. BamHI ends were added to a PCR fragment (nt 9632,231) of CENP-meta ZAP 3-1 subclone by PCR with Pfu polymerase (Stratagene). The PCR product was digested with BamHI, ligated to BamHI digested pQE30, and the resulting plasmid, pQE3-1, was verified by sequencing. Expression constructs were transformed into E. coli strain M15 (QIAGEN). For protein expression, cells were grown to an OD600 of 0.7, fusion protein was induced by the addition of isopropylthiogalactoside (IPTG) to 1 mM, and the cells were grown for an additional 5 h at 37°C. Cells were pelleted, lysed in 8 M urea and the fusion protein purified over a Ni-NTA agarose (QIAGEN) column according to the QIAGEN protocol.
Antibody Production and Affinity Purification
Two rabbit antisera (nos. 6583 and 6584) were raised to an internal peptide sequence of CENP-meta. The peptide, SDKGQQKRRRTWC (aa 385397), was synthesized by Genosys Biotechnologies, Inc., and coupled to KLH via the COOH-terminal cysteine. The KLH-coupled peptide was used to immunize two rabbits at Lampire Biological Laboratories. Antiserum from rabbit 6584 had a significantly high titer against the CENP-meta fusion protein (pQE3-1) and was used for all subsequent work. Antibodies were affinity-purified over a column containing the appropriate fusion protein coupled to cyanogen bromide activated Sepharose (Amersham Pharmacia Biotech), eluting with 200 mM glycine-HCl (pH 2.3) + 0.5 M NaCl. The antibody was neutralized and concentrated into PBS (1.8 mM NaH2PO4, 8.4 mM Na2HPO4, 10 mM KCL, and 150 mM NaCl) using an Ultrafree-15 centrifugal filter (Millipore), stabilized with 40% glycerol, and stored at 4°C.
Cell Culture
Schneider (S2) cells (
Immunofluorescence Microscopy
CENP-meta was immunolocalized in S2 cells attached to coverslips precoated for 5 min at room temperature with 0.1% poly-D-lysine. Nonadherent cells were washed off coverslips in PBS and attached cells were fixed in methanol for 10 min at -20°C. Cells were washed in PBS and incubated for 1 h in blocking buffer (0.2 M glycine, 2.5% fetal bovine serum, and 0.1% Triton X-100, in PBS). Primary antibody incubations were done using 1:100 dilution (14 µg/ml) affinity-purified CENP-meta antisera no. 6584 either alone or in combination with 1:100 dilution of mouse anti-alpha tubulin monoclonal antibody DM1A (Sigma-Aldrich) for 1 h at room temperature. Cells were washed in PBS and incubated in ALEXA-Redconjugated goat antirabbit and/or ALEXA-Greenconjugated goat antimouse secondary antibodies (Molecular Probes) for 1 h at room temperature. Coverslips were washed, incubated with Hoechst 33528 for 2 min, washed again, and mounted in Citrofluor (Ted Pella). Standard fluorescent images were collected using a Princeton Instrument cooled CCD mounted on a Zeiss Axioplan microscope controlled by Metamorph software (Universal Imaging Corp.). Deconvolved images were acquired on a Leica DMRXA/RFA/V automated microscope with a Cooke Sensicam digital camera and processed using Slidebook software (Intelligent Imaging Innovations). Additional image processing was performed using Metamorph (Universal Imaging Corp.) and Adobe Photoshop software.
Whole brains from 3rd instar larvae were fixed and immunostained with monoclonal antibody YL1/2 (1:10 dilution of a culture supernatant; Harlan Bioproducts) to visualize tubulin as described in
Cells visible in "squash" preparations of larval brains were analyzed following protocol 14 of
Immunoprecipitations and Immunoblot Analysis
To prepare extracts for immunoprecipitation (IP), 1.8 x 107 S2 cells per IP were pelleted and washed once in PBS. Cell pellets were then resuspended in 1 ml RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) plus protease inhibitors (0.5 mM AEBSF and 10 µg/ml each aprotinin, pepstatin A, leupeptin, and soybean trypsin inhibitor) and passed 10 times through a 26-gauge needle to shear DNA. The lysate was then incubated on ice for 3 h. To the lysate, 10 µl of affinity-purified (1.4 mg/ml) antibody or 1 µl of pre-immune serum (16 mg/ml) was added, and the lysates were rotated for 2 h at 4°C. 20 µl of Pansorbin beads (Calbiochem-Novabiochem">Calbiochem-Novabiochem) were added and lysates rotated for 2 h at room temperature. Pansorbin beads were spun down and washed 3x in RIPA buffer and then resuspended in 4x sample buffer (50 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.05% bromophenol blue), boiled 10 min, spun 5 min, and loaded onto 10% SDS-PAGE gel. Protein was transferred to nitrocellulose BA83, as previously described (
Online Supplemental Material
Real-time movies were made using manually dechorionated cmet clone embryos in heptane glue. A Bio-Rad MRC1024 laser-scanning confocal microscope was used to collect an image (average of three scans) every 10 s. Files containing real-time series of images were uploaded to NIH image and saved as quicktime movies. Videos 13, further depicting Fig 6 and Fig 7 are at http://www.jcb.org/cgi/content/full/150/1/1/DC1. To ensure a good resolution of the movies, please check that the monitor of your computer is set on millions of colors or true colors (32 bits). The videos show embryos from which images were taken to form Fig 6 and Fig 7. Refer to the respective figure legends for further explanation.
|
|
|
|
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of Drosophila CENP-E Homologues
Degenerate PCR, followed by cDNA and genomic library screening, was used to identify two new Drosophila kinesins (Fig 1 A), both of which map to chromosome 2 at the cytological position 32D-E. We designated these genes CENP-meta (cmet) and CENP-ana (cana). The predicted CENP-meta protein is 2,244 amino acids (257 kD), encoded by a gene comprised of 11 exons and that shares 55 nt of 3' untranslated region (utr) with the 3' utr of the abrupt gene, which is transcribed in the opposite direction (Fig 1 A). The CENP-meta sequence contains a putative nuclear localization sequence (aa 2,1852,190) and a predicted tyrosine phosphorylation site (aa 962-970). The CENP-ana gene encodes a predicted protein of 1,931 aa (222 kD).
The predicted protein structures of both CENP-meta and CENP-ana are similar to those of a variety of kinesins, including CENP-E proteins from human (HCENP-E) and Xenopus (XCENP-E). CENP-meta and CENP-ana both contain an NH2-terminal kinesin-like motor domain linked to a small globular tail domain by a rod domain predicted to form a long discontinuous coiled-coil (Fig 1 B). The proteins encoded by CENP-meta and CENP-ana show considerable sequence similarity throughout their motor and stalk domains (42% identity overall), but they lack any significant sequence similarity to HCENP-E and XCENP-E in their the stalk and tail domains. Nonetheless, a variety of sequence comparison algorithms (ALIGN, NCBI BLAST, GCG PILEUP, and BCM multiple sequence alignment tools, see Materials and Methods) revealed that the motor domains of CENP-meta and CENP-ana are more similar to the motor domains from HCENP-E and XCENP-E than they are to other kinesins (our observations and Case, R., University of California, San Francisco, personal communication). Thus, CENP-meta and CENP-ana appear to be members of the same family of kinesins as HCENP-E and XCENP-E.
CENP-meta Associates with Centromeres/Kinetochores during All Stages of the Cell Cycle
To determine the localization of CENP-meta throughout the cell cycle, a polyclonal antiserum was raised to a 13-mer peptide (SDKGQQKRRRTWC) from the motor domain of CENP-meta (meta peptide aa 385397, Fig 1 A). Affinity-purified serum specifically recognizes and immunoprecipitates two protein species in extracts made from either S2 cells or from whole animals. The major species is a protein of 180 kD (Fig 2 C, lane 2); a second species of
250 kD, which corresponds to the predicted full-length protein constitutes only a minor component (Fig 2 C, lane 2, asterisk). The 250-kD band, in both unfractionated S2 cell lysates and immunoprecipitations, is extremely sensitive to proteolysis. It is only recovered from lysates prepared in the presence of very high concentrations of protease inhibitors and can only be easily detected after concentration of the protein by immunoprecipitation from large cell numbers. Preimmune serum does not recognize these bands in lysates from S2 cells (data not shown) nor does it immunoprecipitate either protein species (Fig 2 C, lane 1). Hence, the 250-kD band is likely to be the full-length CENP-meta protein, which is reduced by proteolysis to the lower molecular mass form. These polypeptides represent CENP-meta, and not CENP-ana, since immunoblotting against a fusion protein that contains the corresponding domain of CENP-ana (aa 230665) reacts with the antibody
140 times more weakly than the analogous fusion protein from CENP-meta (not shown).
In Drosophila S2 tissue culture cells (Fig 2 A), syncytial embryos (Fig 2 B), and squashed larval brain chromosome preparations (Fig 2 D), immunolocalization with affinity-purified antibody revealed that CENP-meta is associated with the centromeric region of chromosomes, presumably kinetochores, during metaphase. This was most apparent in isolated chromosomes (Fig 2 D), where CENP-meta was restricted to the primary constriction of each chromosome pair, a finding consistent with the localization of CENP-E to the corona fibers of the kinetochore (
Examination of cycling cells revealed that CENP-meta is associated with kinetochores during all stages of the cell cycle (Fig 2 A), a behavior that differs from that of HCENP-E and XCENP-E, which have been reported to associate with the kinetochore only upon nuclear envelope breakdown, and then is degraded at the end of mitosis (
A Lethal P-Element Insertional Mutation in Drosophila CENP-meta
Late larval lethal P-element insertions were previously mapped to the area of chromosome 2 around 32D-E ( and cmetlx1. Since CENP-meta and abrupt overlap, we confirmed that cmet
and cmetlx1 fully complement abrupt null mutants. We also found that the lethality of the cmet04431 chromosome can be reverted by precise excision of the l(2)04431 P-element; hence, no other lethal mutations reside on this chromosome.
Blots of total RNA from adult females and embryos of various genotypes were probed for CENP-meta, CENP-ana, and the nearby PPT-2 gene (as well as the mcm5 gene as a loading control). The results demonstrate that cmet is a null allele for CENP-meta at the RNA level (Fig 3, upper panels), but that this allele does not affect the size or abundance of RNAs for CENP-ana (middle panels) or PPT-2 (data not shown).
Null Mutations in CENP-meta Cause Defective Mitosis with Misaligned Chromosomes
To determine the phenotype of mutations in CENP-meta third instar larval brains from animals homozygous or heterozygous for CENP-meta mutations were examined. Analysis of at least 300 fields of fixed, squashed brains from cmetlx1 or cmet animals yielded a striking increase in the fraction of mitotic cells compared with phenotypically wild-type controls (Fig 4 E). While the squashing procedure makes strict interpretation of chromosome alignment difficult, the mitotic cells from mutant animals very frequently contained misaligned chromosomes (Fig 4, AC). These data suggest a role for CENP-meta in chromosome congression, or the maintenance of that congression, in agreement with the model proposed earlier from experiments showing misalignment of chromosomes in spindles assembled in vitro using Xenopus egg extracts depleted of CENP-E (
To ensure that the chromosome positioning defects seen in CENP-meta mutants did not arise from mechanical disruption during squash preparation, and to examine spindle morphology, whole larval brains were examined using indirect immunofluorescence and laser-scanning confocal microscopy. Chromosomes deficient for CENP-meta still apparently bound to bipolar spindles, presumably through kinetochore attachments, but as before, the chromosomes were not as tightly arrayed on a metaphase plate (Fig 4 F) as in wild-type (Fig 4 G).
Using techniques as described above, a second late larval lethal P-element insertion, l(2)00716, was sited in the CENP-ana gene (Fig 1 A). As with embryos carrying mutations in CENP-meta, embryos carrying the P-element displayed an increased mitotic index when fixed, squashed brains were analyzed. However, rather than an increased frequency of prometaphase/metaphase seen with CENP-meta mutants (Fig 4 E), a dramatic increase in anaphase figures was detected (data not shown), suggesting premature anaphase entry or delay during anaphase. Initial efforts to generate CENP-anaspecific antibodies and additional CENP-ana alleles were not successful; more detailed analysis of the functional characteristics of CENP-ana, thus, awaits identification of such reagents.
Germline Deletion Reveals an Early, Essential Role for CENP-meta in Chromosome Alignment
That CENP-meta mutants can survive until pupal stage may reflect a maternal pool of CENP-meta sufficient for early development but that is exhausted during larval cell division. To ascertain whether CENP-meta has an essential role at earlier developmental stages, a strain was constructed in which the CENP-meta product was removed from early embryos as a consequence of cmetlx1 gene disruption in the female germline (referred to hereafter as cmetlx1 mutant embryos). Examination of fixed and stained cmetlx1 embryos revealed that chromosomes failed to align or maintain alignment (Fig 5B and Fig E) and that there were numerous polyploid nuclei as judged by increased intensity of staining with DNA binding dyes. Such defects were observed as early as the eighth embryonic mitosis and became more prominent during later cell cycles in the early Drosophila syncytia. Anaphase defects, such as unequal anaphases lacking centrosomes, were also observed at low frequency (<10% of mitoses). These latter cases were consistent with secondary defects arising from cell cycle progression in the absence of proper chromosome positioning.
Further, as expected for an early mitotic defect of this type (e.g., the dal mutant; and constructed by similar means (see Materials and Methods) was more difficult. Such embryos were produced at much lower frequencies and 20% of these embryos showed defects, such as unusual overall shape, which are strongly suggestive of abnormal oogenesis. However, in the few embryos obtained, prominent chromosome alignment defects could be seen (Fig 5C and Fig F), as well as many nuclei with an abnormally high chromosome content. CP190, usually a marker for centrosomes (Fig 5A and Fig B; arrowed nucleus in Fig 5 C), became redistributed around the chromatin (Fig 5 C, arrowheads).
To examine more directly how the absence of CENP-meta affects chromosome movement, chromosomes in CENP-meta deficient embryos were marked by introducing a stably inherited histone-2A-variant-GFP (
Examination of movies from 16 embryos left no doubt that anaphase onset takes places on schedule, and moreover, it takes place simultaneously for both aligned and misaligned chromosomes. This can be clearly seen for several nuclei where a chromatid pair closer to one pole than the other disjoins contemporaneously with its aligned brethren, and subsequently one chromatid passes across the metaphase plate en route to the more distant pole (e.g., blue or pink pseudo-colored chromosomes; Fig 6A and Fig B). For other nuclei (Fig 6 C), a misaligned chromosome never disjoins, presumably reflecting the failure of one of the kinetochores of the chromosome pair to reassociate successfully with microtubules from the opposite pole. It is probably this mis-segregation event that results in the previously noted aneuploidy and lethality. In no case did the absence of CENP-meta result in premature sister chromatid separation of aligned or misaligned chromosomes and the duration of anaphase was not significantly extended. In cells of wild-type embryos carrying the histone-2A-var-GFP, chromosomes stay aligned at the metaphase plate and sister chromatids segregate cleanly (Fig 6 D).
cmet Embryos Show Earlier Failure of Maintenance of Metaphase Alignment
Consistent with the more severe defects observed with fixed examples of cmet, movies made with this allele revealed defects at an earlier nuclear cycle (in many cases before nuclear migration to the cortex was complete) and a higher proportion of polyploid cells. Again, maintenance of chromosome congression was clearly aberrant (Fig 7; videos further depicting these data available at http://www.jcb.org/cgi/content/full/150/1/1/DC1). For example, in the series of images taken over 300 s in Fig 7 B, tight metaphase alignment of most chromosomes was not maintained, with one chromosome (pseudo-colored green) markedly losing alignment and staying misaligned through initiation of anaphase (at between 150 and 200 s). In this example, upon anaphase onset, both sister chromatids segregated to the same pole. This is also seen in many other examples as illustrated in Fig 7 A (see chromosome pseudo-colored red) and Fig 7 C (see chromosome pseudo-colored orange). Indeed, in the cmet
embryos, examples where misaligned chromatids successfully disjoined and segregated separately at anaphase were much more rare (1 of 12) than for cmetlx1 (7 of 36). Anaphase duration was not detectably extended.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Role of CENP-meta in Chromosome Congression and Movement
The localization of CENP-meta to the kinetochore of chromosomes and the phenotypes of mutants in it suggest that this motor may be a functional homologue of vertebrate CENP-E. That the product of a second gene, CENP-ana, shares a similarly high degree of sequence identity in the motor domain, but no sequence similarity in the tail domains is reminiscent of the bimC family of kinesins, in which members are unambiguously identified by functional analysis, even though some lack similarity in their tail domains. That there are two CENP-Elike proteins in flies may anticipate the situation in mammals. Whereas a single chromosomal locus at 4q24 has been reported for human CENP-E (
To earlier perturbations of CENP-E function, all of which lead to a disruption in chromosome alignment, our real-time observation of mitosis in mutants lacking CENP-meta demonstrates a specific role for CENP-E in maintenance, as opposed to establishment of alignment. Whether CENP-ana has a similar function has not yet been determined; nevertheless, it is readily apparent that the functions of CENP-meta and CENP-ana cannot be identical, since removal of CENP-meta alone causes mitotic disturbance and lethality. What emerges clearly from our data is that multiple motors must be required to generate a stable metaphase alignment since removal of only one, CENP-meta, allows initial congression of many chromosomes, but disturbs its maintenance. In this view, the role of CENP-meta in maintenance of a metaphase plate is to generate a balancing force at the kinetochore, pushing the chromosome away from the pole.
Two additional arguments support the conclusion that CENP-meta is not required to maintain the attachment of microtubules to kinetochores. First, most chromosomes obviously retain bi-oriented attachment to microtubules in the absence of CENP-meta, as seen by the proper disjunction of the majority of chromosomes at anaphase onset. Second, among those chromosomes that do not maintain normal alignment, leaving the metaphase plate abnormally in the absence of CENP-meta, there are several examples where the misaligned chromatid pair disjoins at the normal timing of anaphase with one sister successfully travelling to each pole. Thus, even the misaligned chromosomes in some cases appear to retain (or recover) bipolar attachment, suggesting that failure to remain at the metaphase plate is not solely a result of losing attachment to one of the two poles.
Despite the demonstration of a plus-enddirected, ATP-dependent microtubule motor activity for both an NH2-terminal fragment of CENP-E ( cells can still traverse the entire spindle at anaphase. These chromosomes act as though they not only retained an attachment to the far pole, but that the attachment could properly support, or generate, minus-enddirected movement. Thus, CENP-meta, and by extension CENP-E, may not be essential components of the minus-end force generating mechanism, although they could still play a redundant role with dyneins, MCAK, or other CENP-E relatives such as CENP-ana.
Does CENP-meta have a Role in the Microtubule Checkpoint?
In yeast and vertebrate systems, drugs that cause spindle damage have revealed the presence of a checkpoint that prevents sister chromatid separation when the microtubule spindle is aberrant or missing. The checkpoint is mediated through a diffusible signal of Mad2/Cdc20 released from unattached kinetochores and which inhibits activation of the anaphase promoting complex required for the degradation of regulators of chromatid linkage (e.g., Fang, 1999;
For Drosophila, treatment of dividing Drosophila cells and mitotic syncytial embryos with microtubule depolymerizing agents, such as colchicine and nocodazole, has been reported to yield mitotic arrest with high levels of cyclin. In syncytial embryos the arrest is coordinate, and chromatin is trapped in a prometaphase-like configuration (
![]() |
Footnotes |
---|
The online version of this article contains supplemental material.
Dr. Yucel's current address is Genomica, 1745 38th St., Boulder, CO 80301.
Dr. Philp's current address is CEST, 5 Berners Road, Islington, London N1 0PW, UK.
1 Abbreviations used in this paper: aa, amino acids; nt(s), nucleotide(s); utr, untranslated region.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The authors wish to thank the members (both past and present) of the Cleveland, Goldstein, McIntosh, Winey, and Karpen labs for excellent technical and generous scientific advice. In addition, we wish to thank Harold A. Fisk and Hilary Snaith for their unwavering emotional support and many years of critical scientific discussions.
This work was supported by grant GM29513 to D.W. Cleveland and funds from the Howard Hughes Medical Institute to L.S.B. Goldstein. D.W. Cleveland receives salary support from the Ludwig Institute for Cancer Research. L.S.B. Goldstein is an Investigator of the Howard Hughes Medical Institute. J.K. Yucel was supported by a postdoctoral fellowship from the National Institutes of Health and from the Ludwig Institute for Cancer Research. A.J.V. Philp was supported by a Fulbright Cancer Research Fellowship and by the Howard Hughes Medical Institute.
Submitted: 27 March 2000
Revised: 24 May 2000
Accepted: 2 June 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|