DNA defects target the centrosome
2
© 2000 Macmillan Magazines Ltd news and views E28 NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.nature.com DNA defects target the centrosome Tin Tin Su and Smruti J. Vidwans When cells enter mitosis with DNA that is unfit to be segregated, the consequences appear to be loss of centrosome function, abnormal spindles and a failure to segregate chromosomes. These defects may result from the workings of a surveillance mechanism that acts to cull irreparable nuclei. ntegrity of the genome is paramount in the continuation of species. Accordingly, cells take many steps, when duplicating, to ensure that the genome they pass on to their daughter cells is free from damage. During the replication of many cells, highly con- served checkpoints sense DNA damage or incomplete duplication of the genome, and act to delay the initiation of chromosome seg- regation to the daughter cells (mitosis) 1 . However, some cells may be unable to delay mitosis for a number of reasons, such as being irreversibly committed to mitosis or lacking checkpoint functions. What, then, are the consequences? On page 90 of this issue, Sibon and colleagues address this question by stud- ying embryos of the fruitfly Drosophila melanogaster 2 . They find that entry into mito- sis in the presence of damaged or incom- pletely replicated DNA is accompanied, remarkably, by the inactivation of the micro- tubule-organizing centre or centrosome — one of whose tasks is to generate the microtu- bules of the mitotic spindle, which in turn separates chromosomes. Sibon et al.’s results thus provide evidence for an intriguing link between chromosome defects and the chro- mosome-segregation machinery. The first 13 division cycles of Drosophila melanogaster embryos are nuclear divisions that occur in a common cytoplasm, known as a syncytium. The syncytial cycles consist of alternating S (DNA-replication) phases and mitoses, without intervening gap phases 3 . During the later syncytial cycles, interphase — the portion of the cell cycle between mitoses — lengthens gradually. This lengthening of interphase, or delay to mitotic entry, requires the checkpoint functions encoded by grapes and mei-41, homologues of the fission yeast Chk1 and human ATM/ATR kinases, respectively 4–6 . It has been proposed that the lengthening of interphase in the later syncytial cycles results from a reduced rate of DNA rep- lication, leading to checkpoint-dependent delaying of mitosis 4 . In grp and mei-41 mutant embryos — which lack certain checkpoint functions — syncytial nuclei enter mitosis prematurely and are thought to do so without completing S phase. Likewise, wild-type I nuclei also enter mitosis, albeit with some delay, when DNA replication is blocked, and nuclei with damaged DNA enter mitosis with- out a delay 4,5 . Possibly, checkpoints are relaxed during syncytial cycles, or DNA defects elicit responses other than delaying of mitosis until DNA synthesis or repair has been completed. So, under the right experimental condi- tions, wild-type syncytial nuclei can be induced to enter mitosis with damaged or incompletely replicated DNA. Sibon and colleagues 2 take advantage of these circum- stances, and characterize the mitosis that fol- lows. They report three features of mitosis that occur when the DNA present is unfit for segre- gation — displacement of centrosomal pro- teins that are important for microtubule nucleation, aberrant spindle elaboration, and abnormal chromosome dynamics and segre- gation — in short, a failed mitosis (Fig. 1) 2 . Re- entry into interphase ensues, resulting in nuclei with an incorrect number of chromo- somes (aneuploid nuclei). Thus, it appears that the state of the DNA at mitosis plays a part in the proper execution of mitosis, raising a number of interesting questions. Does mitosis fail simply because the DNA cannot be segre- gated, or is the process of mitosis aborted? If these mitoses are indeed aborted, what are the mechanisms responsible and what purpose does this serve the cell and the overall develop- ment of an organism? Are such effects observed in other cell types and organisms? In trying to make sense of these observa- tions, it is worth considering how DNA defects might result in mitotic defects, and whether these mitotic errors are independent consequences of entry into mitosis with DNA lesions, or whether one mitotic defect leads to another. It is possible that faults in lining up chromosomes to segregate them into the two daughter cells (chromosome congression), and defects in segregation itself, are due to physical constraints imposed by aberrant DNA, such as the absence of contiguous DNA strands or the presence of replication inter- mediates. Furthermore, in preparation for mitosis, chromosomes become decorated with proteins such as the condensins (which have a role in the dramatic condensation of chromosomes observed at mitosis) and cohesins (which mediate the cohesion of sis- ter chromosomes needed to keep them together until they head for different daughter cells) 7,8 . Damaged or incompletely replicated DNA may not acquire such proteins properly, thus contributing to chromosome-congres- sion and chromosome-segregation failures. So, some of the observed defects might indeed be the direct result of DNA defects. In addition to the defects in chromo- somes, the centrosomes lose three compo- nents of their γ-tubulin-based ‘ring complex’, and so appear to be impaired in nucleation of microtubules 2 . Consistent with this idea, the resulting spindles, although bipolar as nor- mal, show unfocused ends and lack astral microtubules (the spindles are therefore Figure 1 The response of Drosophila syncytial nuclei to DNA defects. Sibon and colleagues 2 used three experimental protocols (the use of embryos with mutations in so- called checkpoint proteins, a DNA-synthesis inhibitor, or laser damage to DNA) to produce mitotic entry in the presence of damaged or incompletely replicated DNA. Characterization of the ensuing mitoses revealed centrosome and spindle defects. Chromosomes also failed to align and segregate. Exit from mitosis followed eventually, resulting in aneuploid nuclei with an incorrect complement of chromosomes. Spindle defects Anastral Unfocused poles Chromosome defects Failure to congress on metaphase plate Failure to segregate Centrosome defects Loss of γ-tubulin ring complex components ? Entry into mitosis with damaged or incompletely replicated DNA Checkpoint mutations DNA-synthesis inhibitor Photo-damage
Transcript of DNA defects target the centrosome
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E28 NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.nature.com
DNA defects target the centrosome
Tin Tin Su and Smruti J. Vidwans
When cells enter mitosis with DNA that is unfit to be segregated, the consequences appear to be loss of centrosome function, abnormal spindles and a failure to segregate chromosomes. These defects may result from the workings of a surveillance mechanism that acts to cull irreparable nuclei.
ntegrity of the genome is paramount in thecontinuation of species. Accordingly, cellstake many steps, when duplicating, to
ensure that the genome they pass on to theirdaughter cells is free from damage. Duringthe replication of many cells, highly con-served checkpoints sense DNA damage orincomplete duplication of the genome, andact to delay the initiation of chromosome seg-regation to the daughter cells (mitosis)1.However, some cells may be unable to delaymitosis for a number of reasons, such as beingirreversibly committed to mitosis or lackingcheckpoint functions. What, then, are theconsequences? On page 90 of this issue, Sibonand colleagues address this question by stud-ying embryos of the fruitfly Drosophilamelanogaster2. They find that entry into mito-sis in the presence of damaged or incom-pletely replicated DNA is accompanied,remarkably, by the inactivation of the micro-tubule-organizing centre or centrosome —one of whose tasks is to generate the microtu-bules of the mitotic spindle, which in turnseparates chromosomes. Sibon et al.’s results
thus provide evidence for an intriguing linkbetween chromosome defects and the chro-mosome-segregation machinery.
The first 13 division cycles of Drosophilamelanogaster embryos are nuclear divisionsthat occur in a common cytoplasm, known asa syncytium. The syncytial cycles consist ofalternating S (DNA-replication) phases andmitoses, without intervening gap phases3.During the later syncytial cycles, interphase —the portion of the cell cycle between mitoses— lengthens gradually. This lengthening ofinterphase, or delay to mitotic entry, requiresthe checkpoint functions encoded by grapesand mei-41, homologues of the fission yeastChk1 and human ATM/ATR kinases,respectively4–6. It has been proposed that thelengthening of interphase in the later syncytialcycles results from a reduced rate of DNA rep-lication, leading to checkpoint-dependentdelaying of mitosis4. In grp and mei-41 mutantembryos — which lack certain checkpointfunctions — syncytial nuclei enter mitosisprematurely and are thought to do so withoutcompleting S phase. Likewise, wild-type
I
nuclei also enter mitosis, albeit with somedelay, when DNA replication is blocked, andnuclei with damaged DNA enter mitosis with-out a delay4,5. Possibly, checkpoints are relaxedduring syncytial cycles, or DNA defects elicitresponses other than delaying of mitosis untilDNA synthesis or repair has been completed.
So, under the right experimental condi-tions, wild-type syncytial nuclei can beinduced to enter mitosis with damaged orincompletely replicated DNA. Sibon andcolleagues2 take advantage of these circum-stances, and characterize the mitosis that fol-lows. They report three features of mitosis thatoccur when the DNA present is unfit for segre-gation — displacement of centrosomal pro-teins that are important for microtubulenucleation, aberrant spindle elaboration, andabnormal chromosome dynamics and segre-gation — in short, a failed mitosis (Fig. 1)2. Re-entry into interphase ensues, resulting innuclei with an incorrect number of chromo-somes (aneuploid nuclei). Thus, it appearsthat the state of the DNA at mitosis plays a partin the proper execution of mitosis, raising anumber of interesting questions. Does mitosisfail simply because the DNA cannot be segre-gated, or is the process of mitosis aborted? Ifthese mitoses are indeed aborted, what are themechanisms responsible and what purposedoes this serve the cell and the overall develop-ment of an organism? Are such effectsobserved in other cell types and organisms?
In trying to make sense of these observa-tions, it is worth considering how DNAdefects might result in mitotic defects, andwhether these mitotic errors are independentconsequences of entry into mitosis with DNAlesions, or whether one mitotic defect leads toanother. It is possible that faults in lining upchromosomes to segregate them into the twodaughter cells (chromosome congression),and defects in segregation itself, are due tophysical constraints imposed by aberrantDNA, such as the absence of contiguous DNAstrands or the presence of replication inter-mediates. Furthermore, in preparation formitosis, chromosomes become decoratedwith proteins such as the condensins (whichhave a role in the dramatic condensation ofchromosomes observed at mitosis) andcohesins (which mediate the cohesion of sis-ter chromosomes needed to keep themtogether until they head for different daughtercells)7,8. Damaged or incompletely replicatedDNA may not acquire such proteins properly,thus contributing to chromosome-congres-sion and chromosome-segregation failures.So, some of the observed defects might indeedbe the direct result of DNA defects.
In addition to the defects in chromo-somes, the centrosomes lose three compo-nents of their γ-tubulin-based ‘ring complex’,and so appear to be impaired in nucleation ofmicrotubules2. Consistent with this idea, theresulting spindles, although bipolar as nor-mal, show unfocused ends and lack astralmicrotubules (the spindles are therefore
Figure 1 The response of Drosophila syncytial nuclei to DNA defects. Sibon and colleagues2 used three experimental protocols (the use of embryos with mutations in so-called checkpoint proteins, a DNA-synthesis inhibitor, or laser damage to DNA) to produce mitotic entry in the presence of damaged or incompletely replicated DNA. Characterization of the ensuing mitoses revealed centrosome and spindle defects. Chromosomes also failed to align and segregate. Exit from mitosis followed eventually, resulting in aneuploid nuclei with an incorrect complement of chromosomes.
Spindle defectsAnastralUnfocused poles
Chromosome defectsFailure to congress onmetaphase plateFailure to segregate
Centrosome defectsLoss of γ-tubulin ringcomplex components
?
Entry into mitosiswith damagedor incompletelyreplicated DNA
Checkpointmutations
DNA-synthesisinhibitor
Photo-damage
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NATURE CELL BIOLOGY | VOL 2 | FEBRUARY 2000 | cellbio.nature.com E29
called ‘anastral’). On the basis of these spin-dles, the authors propose a model in whichthe impairment of centrosomes leads toabnormal spindle elaboration and, conse-quently, a failure to align and segregate chro-mosomes. Although this model is attractive,it is not clear to what extent anastral spindlesreally are impaired in chromosome segrega-tion. Centrosomes have an instrumental rolein the elaboration of the mitotic spindle, buttheir absolute requirement in the executionof mitosis is controversial. Mitosis (and mei-osis) progress in various normal cell typesand mutants with compromised or missingcanonical centrosomes9. So, the extent of thecontribution of centrosomal defects to thechromosome dynamics observed by Sibon etal. remains unknown, and it remains possiblethat both centrosome-inactivation and DNAdefects contribute to the abnormal chromo-some dynamics.
Despite these uncertainties, the observa-tion that centrosome function is compro-mised is intriguing, because it is notimmediately obvious why or how lesions inDNA might affect centrosome behaviour.Available data help to rule out the involve-ment of a few pathways and molecules. Asembryos with mutations in the grp and mei-41 checkpoint genes also show impaired cen-trosome function, these genes are clearly dis-pensable for centrosome inactivation. Thearrangement of the kinetochore regions ofchromosomes (where the spindle attaches)appears normal, suggesting that the spindlecheckpoint (which monitors these attach-ments) is not a player2. It is possible thatother, as yet uncovered, checkpoint functionsare required (possibly involving a Drosophilahomologue of Cds1/Chk2 or another ATM/ATR homologue). Alternatively, a newcheckpoint pathway may be at work here,sensing mitotic DNA defects and signalling tothe centrosome.
What, then, might cells achieve by impair-ing centrosome function during mitosis whenDNA is faulty? A possible clue comes fromlooking at the fate of nuclei that undergomitosis with defective DNA. In previous stud-ies of syncytial embryos that had been exposedto radiation or DNA-synthesis inhibitors,nuclei left the embryonic cortex after mitosisand sank into the interior yolk mass, leavingtheir centrosomes behind10,11. This nuclearexile has been proposed to act to cull damagednuclei, ensuring the integrity of the cells con-stituting the embryo — and so representingan embryonic equivalent to apoptosis (pro-grammed cell death)12. An attractive possibil-ity is that defects in centrosome function inmitosis somehow contribute to the detach-ment of nuclei from their centrosomes andeventual sinking. Although this phenomenonis observed in wild-type embryos with sus-tained DNA defects, it is not observed inembryos with mutations in grapes and mei-41(refs 5, 6). In these mutants, nuclei remain onthe surface as clusters of daughter nuclei that
underwent failed mitoses, which might pointto a role for these checkpoint genes in cullingof damaged nuclei.
Are these effects unique to the special envi-ronment of early Drosophila embryos? Thereare significant differences between cell-cycleregulation in early embryos of insects andamphibians and that of later somatic cycles.For example, cell cycles in the early embryoslack gap phases and may have more relaxedcheckpoint controls3. And, in the case of Dro-sophila syncytial cycles, all of the nuclei (withthe exception of those in the future germ cells)share a common cytoplasm. Thus, theprocessing of a damaged nucleus under thesecircumstances might differ from how a uni-cellular organism or a vertebrate somatic celldeals with defective DNA. Yet several lines ofevidence indicate that the means and ends tocell-cycle control in response to DNA defectsmay be conserved in different cell types.
Generally speaking, mitosis (or meiosis)may be arrested either before it begins orbefore chromosomes segregate (Fig. 2)1,13.Interestingly, when the premeiosis arrest isdisabled in fission yeast, a chromosome-seg-regation arrest point becomes activated, indi-cating that some cell divisions may beregulated at both arrest points in response tocheckpoint activation13. The situation in Dro-sophila syncytial cycles may be similar — thatis, if the delay in entry into mitosis is not exe-cuted or maintained, then chromosome seg-regation may become blocked2,5. Moreover,Sibon et al.’s results2 indicate that thisresponse may involve and rely on centrosomeinactivation and spindle flaws. It is possiblethat a similar response exists in culturedhuman cells. The primary response to DNAdamage here is a delay in mitotic entry, andwhen this response is either disabled or notmaintained, cells enter mitosis1,14,15. Thesecells, however, do not complete mitosis or cell
division, become aneuploid and, in somecases, are targeted for apoptosis. Although thismitotic failure may simply result frommechanical difficulties in separating damagedchromosomes, it is also possible that in mam-malian cells, like in Drosophila, a mechanismis at work that monitors mitotic DNA defectsand can abort mitosis. Assays of centrosomeinactivation, which has no intuitively obviouslink to DNA defects, and of the dependence ofmitotic failure on known checkpoint func-tions may be ways to look for this new, andpotentially universal, connection between theDNA checkpoint and the mitotic spindle. hTin Tin Su is in the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.e-mail: [email protected] J. Vidwans is a Howard Hughes Medical Institute Predoctoral Fellow in the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA.
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Nature Cell Biol. 2, 90–95 (2000).
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12. Sullivan, W., Daily, D. R., Fogarty, P., Yook, K. J. & Pimpinelli,
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Figure 2 Damaged or incompletely replicated DNA can arrest either the entry into mitosis or chromosome segregation during mitosis, depending on cell type. Some cell cycles, such as meiosis in fission yeast, may use both arrest points; if the first arrest point is disabled and cells enter meiosis with incompletely replicated DNA, the second arrest point is enforced and chromosome segregation does not occur. Drosophila syncytial divisions may also be regulated in this way.
DNA defectsDrosophilasyncytial mitosis Budding yeast
mitosis
Drosophilasyncytialmitosis
Fission yeastmitosis
Vertebratemitosis
Fission yeastmeiosis
Fission yeastmeiosis
Entry into mitosis(or meiosis)
Chromosome segregation
