BACKGROUND
centrioles are cylindrical structures located within the core of the centrosome.
although the localization of centrioles within the centrosome together with their precise duplication prior to mitosis has suggested a role in bipolar spindle assembly or function, the actual role of centrioles in cell division remains unclear and controversial.
cells from which centrioles and centrosomes are ablated can still form bipolar spindles via a centrosome-independent self-organization process, but the effectiveness of such spindles in terms of chromosome segregation has not been carefully measured.
based on the circumstantial evidence that tumor cells which display genomic instability also frequently show aberrations in centriole structure or copy number  <cit> , it has been proposed that centrioles may participate in the maintenance of genomic stability.
if centrioles play a role in chromosome segregation, then mutations in genes encoding centriolar proteins would be expected to result in genomic instability.
the ef-hand protein centrin  <cit>  is one of the few identified centriolar proteins.
centrin is found in all eukaryotes, and is localized to centrioles in vertebrate cells  <cit> , basal bodies in green algae  <cit> , and spindle pole bodies  in yeast  <cit> .
centrin is functionally required for centriole duplication  <cit>  and inheritance  <cit> .
rnai experiments in mammalian and algal cells have indicated that reduction of centrin can result in cell division arrest  <cit>  and cytokinesis failure  <cit> .
however, it is unclear if these defects are indirectly caused by abnormal centriole structure or copy number, or if instead they represent pleiotropic effects due to the direct participation of centrin in the cell division process.
centrin also appears to be involved in many other cellular processes, including flagellar excision  <cit> , nuclear mrna export  <cit> , cell membrane integrity  <cit>  and homologous recombination  <cit> .
consistent with these multiple roles, centrin localization is not restricted to centrioles  <cit> , and centrin has been identified as a component of nuclear pores and flagellar dynein complexes  <cit> .
centrin is thus a versatile molecule that has been adapted to perform multiple diverse cellular functions.
given the fact that centrin localizes to centrioles, which are potentially involved in genome maintenance, together with the fact that centrin is functionally involved in centriole duplication and segregation, an obvious question is whether centrin plays a role in genomic stability.
in this report we address this question using a centrin mutation in the green alga chlamydomonas reinhardtii.
RESULTS
genomic instability in the centrin mutant vfl2-1
we tested whether centrin plays a role in genomic instability using a genetic assay for loss of heterozygosity in chlamydomonas reinhardtii.
chlamydomonas  is a unicellular green alga with genetics similar to yeast, but which, unlike yeast, has centrioles  that are virtually identical to centrioles of vertebrate cells.
in chlamydomonas, centrin polymerizes into long fibers that link the centrioles/basal bodies to the nucleus.
these fibers are called the nucleus basal body connectors  or rhizoplasts  <cit> .
the vfl2- <dig> mutant has a point mutation in the centrin gene  <cit> , and these mutants produce a reduced quantity of centrin protein that is unable to polymerize into a rhizoplast.
the vfl <dig> mutation appears to result in a defect in centriole segregation, because mutant cells have a variable number of centrioles .
despite their centriole number variation, these mutants are viable and form seemingly normal bipolar mitotic spindles.
it has been argued on this basis that centrin-based rhizoplasts do not play a role in mitosis  <cit> .
importantly, the centrioles in a vfl <dig> mutant cell are not located at the spindle poles  <cit> , consistent with ultrastructural studies showing that, in green algae, centrioles are joined to the spindle poles by centrin-based fibers derived from the rhizoplast  <cit> .
the high viability of vfl <dig> mutants thus allows us to measure genomic instability using genetic assays because populations of mutant cells can be grown for a sufficient number of generations to allow rare chromosome loss events to be detected.
we measured genomic instability in vfl <dig> mutants using a simple genetic assay for loss of heterozygosity  previously developed by dutcher and coworkers  <cit> .
this assay uses the act <dig> mutation, which confers recessive resistance to cycloheximide  <cit> .
the act <dig> heterozygous diploids are sensitive to cycloheximide, while homozygous or hemizygous act <dig> diploids are cycloheximide-resistant.
when a diploid culture is plated on media containing cycloheximide, only cells that have lost the wild-type act <dig> allele can grow.
by measuring the frequency with which such cells arise, the rate of chromosome loss can be calculated .
as illustrated in fig.
<dig>  using  <dig> independently constructed diploid lines each for wild type and vfl <dig>  we found that the loh rate per cell division increased from  <dig>  × 10- <dig> ±  <dig>  × 10- <dig> sem  in wild type, to  <dig>  × 10- <dig> ±  <dig>  × 10- <dig> sem  in vfl <dig> mutants.
this difference is statistically significant .
the increase in loss of heterozygosity in the vfl <dig> centrin mutant relative to wild-type cells indicates that centrin plays a role in genomic stability.
genomic instability occurs via chromosome loss
in principle the loh seen in vfl <dig> mutants could occur either by increased recombination or increased chromosome loss .
the latter would be consistent with a role for centrin or centrioles in mitotic spindle function, while an effect on recombination would suggest a spindle-independent role in dna maintenance or chromosome dynamics.
it is critical to distinguish these pathways because a role for centrin in dna recombination and repair has been documented in plants  <cit> .
given the close phylogenetic relation between green algae like chlamydomonas and green plants, the involvement of centrin in recombination in plants raises the real possibility that the loss of heterozygosity observed in vfl <dig> might have nothing to do with mitotic spindle function and might instead be driven by increased recombination.
in order to determine whether genomic instability in the vfl <dig> mutant is caused by increased recombination or chromosome loss, we distinguished between the two pathways by analyzing the mating type  locus, which is located on the opposite arm of the same chromosome from act <dig> .
diploid cells are heterozygous at mt and contain both the mt+ and mt- alleles.
because the act <dig> and mt loci are on opposite arms of the same chromosome, and recombination only causes loh on a single arm distal to the crossover site, it can be inferred that only chromosome loss, and not mitotic recombination, could lead to the simultaneous loss of both the wild-type act <dig> gene and the mt+ allele .
we therefore tested lines that had undergone loh at the act <dig> locus for chromosome loss versus recombination, by analyzing the mt locus using pcr primers specific for the mt- and mt+ alleles  <cit> .
as illustrated in fig.
<dig>  prior to selection on cycloheximide, all wild-type and vfl2/vfl <dig> diploid lines were indeed diploid, as judged by the fact that they were heterozygous at the mt locus and contained both mt+ and mt- alleles as expected for a diploid.
in wild type, of  <dig> independently constructed diploid lines selected on cycloheximide, only two lines showed concomitant loss of the mt+ allele and, therefore, chromosome loss.
in contrast, of  <dig> cycloheximide resistant vfl <dig> lines, we found that  <dig> had also lost the mt+ allele, loss of which indicated that the lines had undergone chromosome loss.
these chromosome loss events apparently represent the loss of individual chromosomes rather than an overall return to haploidy, as the cells were able to grow on media lacking arginine and thus seemed to have retained the two homologs of the chromosome containing the arg2/arg <dig> mutations used to select for diploidy .
the difference in the proportion of loh events due to chromosome loss  was highly significant .
multiplying the loh rate by the fraction of loh events due to chromosome loss, we found that the total rate of chromosome loss per cell division increased almost 100-fold, from  <dig>  × 10- <dig> in wild type to  <dig>  × 10- <dig> in vfl <dig> mutants.
cell death during g <dig> in vfl <dig> mutants
the fact that vfl <dig> mutants show increased chromosome loss in the genetic assay described above leads to the prediction that haploid vfl <dig> cells should frequently produce inviable progeny cells.
this prediction follows from the fact that all chlamydomonas chromosomes contain essential genes, so a single chromosome loss event for any of the  <dig> chromosomes should produce an inviable progeny cell when haploid cell division are analyzed.
consistent with this prediction, analysis of cell viability using the vital stain phenosaffranin indicated that vfl <dig> cultures contain a higher fraction of inviable cells than wild type .
in order to test this prediction more quantitatively, we examined the outcomes of individual cell division events.
synchronized cultures of haploid chlamydomonas cells were embedded in agarose pads under a coverslip and imaged during g <dig>  by phase contrast microscopy as previously described  <cit> .
viable cells were identified and their position marked, and then their progeny were examined during the first g <dig> phase after division.
only cells that divided successfully to give two or four progeny were scored .
as a test of the methodology, we compared outcomes of division for wild-type cells and fla10- <dig> mutant cells grown at 21°c.
the fla10- <dig> mutation occurs in a kinesin involved in flagellar assembly, and it has been reported that fla10- <dig> mutants show an increased rate of mitotic chromosome loss when grown at the permissive temperature  <cit> .
if chromosome loss leads to increased production of inviable progeny, then this should be detectable in our analysis.
indeed, as shown in fig.
<dig>  fla10- <dig> cells showed a significant increase in the fraction of divisions producing inviable progeny.
we then applied this method to vfl <dig> mutant cells.
as indicated in fig.
<dig>  we found that vfl <dig> showed a similar level of inviable progeny production as was observed in fla <dig> and significantly higher than in wild type.
the cell death was observed during the first g <dig> after division and therefore did not result from a prolonged arrest or from a failure during subsequent s phase or mitosis.
dead cells were often roughly similar in size to their live siblings, suggesting that they were initially able to grow but eventually became inviable as they grew.
in most cases the viable sibling cells retained flagella, indicating the culture had not yet reached the first s phase , confirming that death occurred during g <dig>  this cell death during g <dig> is consistent with increased chromosome loss during mitosis, which should result in death during growth as essential gene products become depleted.
we cannot, however, rule out the possibility that the cell death results from other defects in cell division.
in order to test whether supernumerary centrioles might cause the elevated cell death we observed, we asked whether the cell division defects in vfl <dig> correlated with the number of centrioles present.
as previously shown, the fact that all centrioles are active as basal bodies in vfl <dig> mutants allows the number of centrioles to be inferred from the number of flagella  <cit> .
we therefore examined the outcome of cell divisions of cells having either the correct centriole copy number , too few centrioles  or supernumerary centrioles .
as indicated in fig.
<dig>  the fractions of inviable progeny were statistically similar for all three classes.
we especially note that the cells with supernumerary centrioles did not show an increase in cell death after division.
discussion
these results demonstrate that the vfl2- <dig> point mutation  <cit>  in centrin leads to increased genomic instability via an increase in chromosome loss, suggesting that centrin plays a role in mitotic chromosome segregation.
formally, these results suggest that the gene encoding centrin could act as a "housekeeper" type tumor suppressor gene, which when mutated would predispose a cell to tumorigenesis indirectly by increasing genomic instability.
the mechanism by which the vfl <dig> mutation leads to increased chromosome loss is unknown.
the chromosome missegregation seen in the vfl <dig> centrin mutant is reminiscent of the ipl  phenotype seen in yeast mutants defective in the yeast centrin homolog cdc <dig>  <cit> , which is presumably due to a failure to form a mitotic spindle.
by analogy with cdc <dig> mutants, it is then possible that the chromosome loss in vfl <dig> results from defective spindles.
however, unlike in yeast where the spindle pole body is absolutely necessary to form a mitotic spindle, in higher eukaryotes it has been shown that bipolar spindles can self-assemble in the absence of centrioles or centrosomes  <cit> .
thus it was not obvious a priori that a defect in a centriole-associated protein  should have any effect on mitosis.
indeed, in the specific case of the chlamydomonas vfl <dig> mutant, it has previously been reported that even though the vfl <dig> mutation causes centrioles to detach from the spindle poles, the acentriolar spindles are nonetheless still bipolar  <cit> .
on this basis it was concluded by wright and coworkers  <cit>  that centrin does not play a role in mitosis.
it might therefore seem surprising that we find an increase in chromosome loss in the vfl <dig> mutant.
however, while the ability of cells lacking centrioles or centrosomes to form bipolar spindles is well documented, and these spindles can be seen to undergo an apparently normal anaphase chromosome separation  <cit> , the question of whether such acentriolar spindles might have an altered fidelity of chromosome segregation compared to normal spindles has never been directly addressed.
we speculate that the chromosome missegregation we observe in the vfl <dig> mutant reflects a decreased effectiveness of acentriolar spindles that self-assemble in this mutant.
this would suggest a mitotic fidelity function for centrioles, which requires not merely the presence of centrioles in the cell, but also their specific localization at the spindle pole.
alternatively, the chromosome loss we observe could be a byproduct of multipolar spindles.
a small fraction  of vfl <dig> cells have three or more centrioles that could in theory give rise to tripolar or tetrapolar spindles.
we suspect this is not the case because only bipolar spindles have been observed in these mutants  <cit> .
moreover, since centrioles are not located at spindle poles in this mutant, it is unlikely that the number of centrioles would have any effect on the number of spindle poles.
the lack of centrioles at the poles also rules out configurations such as those seen during mitosis in multinucleate ptk <dig> cells  <cit> , when multiple centriole pairs line up on either side of a single metaphase plate to form a masked multipolar spindle.
furthermore, to the extent that production of inviable cells during mitosis reflects chromosome loss, the data of fig.
<dig> argue against multipolar spindles playing a dominant role in chromosome loss since cells containing supernumerary centrioles do not show any increase in the rate of production of inviable progeny cells.
it is also possible that the chromosome loss in vfl <dig> mutants arises through a spindle-independent mechanism.
because centrin may be involved in nuclear transport  <cit> , it is possible that the vfl <dig> mutation could cause defects in chromosome organization due to a nuclear trafficking defect, leading indirectly to chromosome loss.
we also note that disruption of centrin by rnai in chlamydomonas has been reported to cause cytokinesis defects leading to binucleate cells  <cit> .
however such a cytokinesis defect cannot explain our results because the loh assay we employed selects for loss of act <dig>  rather than the presence of additional copies.
detailed cell-biological analysis of centrin mutant cells during division will be necessary to understand the basis of the chromosome loss we have observed.
CONCLUSIONS
we have found that a mutation in the centriole-associated protein centrin, which causes affected cells to form acentriolar spindles and lack centrin-based fibers, produces an increase in genomic instability caused by an increase in the rate of chromosome loss.
these results indicate that either centrioles or the centrin protein itself are involved in increasing the fidelity of mitotic chromosome segregation.

 centrioles are cylindrical structures located within the core of the centrosome. although the localization of centrioles within the centrosome together with their precise duplication prior to mitosis has suggested a role in bipolar spindle assembly or function, the actual role of centrioles in cell division remains unclear and controversial. cells from which centrioles and centrosomes are ablated can still form bipolar spindles via a centrosome-independent self-organization process, but the effectiveness of such spindles in terms of chromosome segregation has not been carefully measured. based on the circumstantial evidence that tumor cells which display genomic instability also frequently show aberrations in centriole structure or copy number, it has been proposed that centrioles may participate in the maintenance of genomic stability. if centrioles play a role in chromosome segregation, then mutations in genes encoding centriolar proteins would be expected to result in genomic instability. the ef-hand protein centrin is one of the few identified centriolar proteins. centrin is found in all eukaryotes, and is localized to centrioles in vertebrate cells, basal bodies in green algae, and spindle pole bodies in yeast. centrin is functionally required for centriole duplication and inheritance. rnai experiments in mammalian and algal cells have indicated that reduction of centrin can result in cell division arrest and cytokinesis failure. however, it is unclear if these defects are indirectly caused by abnormal centriole structure or copy number, or if instead they represent pleiotropic effects due to the direct participation of centrin in the cell division process. centrin also appears to be involved in many other cellular processes, including flagellar excision, nuclear mrna export, cell membrane integrity and homologous recombination. consistent with these multiple roles, centrin localization is not restricted to centrioles, and centrin has been identified as a component of nuclear pores and flagellar dynein complexes. centrin is thus a versatile molecule that has been adapted to perform multiple diverse cellular functions. given the fact that centrin localizes to centrioles, which are potentially involved in genome maintenance, together with the fact that centrin is functionally involved in centriole duplication and segregation, an obvious question is whether centrin plays a role in genomic stability. in this report we address this question using a centrin mutation in the green alga chlamydomonas reinhardtii.  genomic instability in the centrin mutant vfl2-1 we tested whether centrin plays a role in genomic instability using a genetic assay for loss of heterozygosity in chlamydomonas reinhardtii. chlamydomonas is a unicellular green alga with genetics similar to yeast, but which, unlike yeast, has centrioles that are virtually identical to centrioles of vertebrate cells. in chlamydomonas, centrin polymerizes into long fibers that link the centrioles/basal bodies to the nucleus. these fibers are called the nucleus basal body connectors or rhizoplasts. the vfl2- mutant has a point mutation in the centrin gene, and these mutants produce a reduced quantity of centrin protein that is unable to polymerize into a rhizoplast. the vfl mutation appears to result in a defect in centriole segregation, because mutant cells have a variable number of centrioles. despite their centriole number variation, these mutants are viable and form seemingly normal bipolar mitotic spindles. it has been argued on this basis that centrin-based rhizoplasts do not play a role in mitosis. importantly, the centrioles in a vfl mutant cell are not located at the spindle poles, consistent with ultrastructural studies showing that, in green algae, centrioles are joined to the spindle poles by centrin-based fibers derived from the rhizoplast. the high viability of vfl mutants thus allows us to measure genomic instability using genetic assays because populations of mutant cells can be grown for a sufficient number of generations to allow rare chromosome loss events to be detected. we measured genomic instability in vfl mutants using a simple genetic assay for loss of heterozygosity previously developed by dutcher and coworkers. this assay uses the act mutation, which confers recessive resistance to cycloheximide. the act heterozygous diploids are sensitive to cycloheximide, while homozygous or hemizygous act diploids are cycloheximide-resistant. when a diploid culture is plated on media containing cycloheximide, only cells that have lost the wild-type act allele can grow. by measuring the frequency with which such cells arise, the rate of chromosome loss can be calculated. as illustrated in fig. using independently constructed diploid lines each for wild type and vfl we found that the loh rate per cell division increased from × 10- ± × 10- sem in wild type, to × 10- ± × 10- sem in vfl mutants. this difference is statistically significant. the increase in loss of heterozygosity in the vfl centrin mutant relative to wild-type cells indicates that centrin plays a role in genomic stability. genomic instability occurs via chromosome loss in principle the loh seen in vfl mutants could occur either by increased recombination or increased chromosome loss. the latter would be consistent with a role for centrin or centrioles in mitotic spindle function, while an effect on recombination would suggest a spindle-independent role in dna maintenance or chromosome dynamics. it is critical to distinguish these pathways because a role for centrin in dna recombination and repair has been documented in plants. given the close phylogenetic relation between green algae like chlamydomonas and green plants, the involvement of centrin in recombination in plants raises the real possibility that the loss of heterozygosity observed in vfl might have nothing to do with mitotic spindle function and might instead be driven by increased recombination. in order to determine whether genomic instability in the vfl mutant is caused by increased recombination or chromosome loss, we distinguished between the two pathways by analyzing the mating type locus, which is located on the opposite arm of the same chromosome from act. diploid cells are heterozygous at mt and contain both the mt+ and mt- alleles. because the act and mt loci are on opposite arms of the same chromosome, and recombination only causes loh on a single arm distal to the crossover site, it can be inferred that only chromosome loss, and not mitotic recombination, could lead to the simultaneous loss of both the wild-type act gene and the mt+ allele. we therefore tested lines that had undergone loh at the act locus for chromosome loss versus recombination, by analyzing the mt locus using pcr primers specific for the mt- and mt+ alleles. as illustrated in fig. prior to selection on cycloheximide, all wild-type and vfl2/vfl diploid lines were indeed diploid, as judged by the fact that they were heterozygous at the mt locus and contained both mt+ and mt- alleles as expected for a diploid. in wild type, of independently constructed diploid lines selected on cycloheximide, only two lines showed concomitant loss of the mt+ allele and, therefore, chromosome loss. in contrast, of cycloheximide resistant vfl lines, we found that had also lost the mt+ allele, loss of which indicated that the lines had undergone chromosome loss. these chromosome loss events apparently represent the loss of individual chromosomes rather than an overall return to haploidy, as the cells were able to grow on media lacking arginine and thus seemed to have retained the two homologs of the chromosome containing the arg2/arg mutations used to select for diploidy. the difference in the proportion of loh events due to chromosome loss was highly significant. multiplying the loh rate by the fraction of loh events due to chromosome loss, we found that the total rate of chromosome loss per cell division increased almost 100-fold, from × 10- in wild type to × 10- in vfl mutants. cell death during g in vfl mutants the fact that vfl mutants show increased chromosome loss in the genetic assay described above leads to the prediction that haploid vfl cells should frequently produce inviable progeny cells. this prediction follows from the fact that all chlamydomonas chromosomes contain essential genes, so a single chromosome loss event for any of the chromosomes should produce an inviable progeny cell when haploid cell division are analyzed. consistent with this prediction, analysis of cell viability using the vital stain phenosaffranin indicated that vfl cultures contain a higher fraction of inviable cells than wild type. in order to test this prediction more quantitatively, we examined the outcomes of individual cell division events. synchronized cultures of haploid chlamydomonas cells were embedded in agarose pads under a coverslip and imaged during g by phase contrast microscopy as previously described. viable cells were identified and their position marked, and then their progeny were examined during the first g phase after division. only cells that divided successfully to give two or four progeny were scored. as a test of the methodology, we compared outcomes of division for wild-type cells and fla10- mutant cells grown at 21°c. the fla10- mutation occurs in a kinesin involved in flagellar assembly, and it has been reported that fla10- mutants show an increased rate of mitotic chromosome loss when grown at the permissive temperature. if chromosome loss leads to increased production of inviable progeny, then this should be detectable in our analysis. indeed, as shown in fig. fla10- cells showed a significant increase in the fraction of divisions producing inviable progeny. we then applied this method to vfl mutant cells. as indicated in fig. we found that vfl showed a similar level of inviable progeny production as was observed in fla and significantly higher than in wild type. the cell death was observed during the first g after division and therefore did not result from a prolonged arrest or from a failure during subsequent s phase or mitosis. dead cells were often roughly similar in size to their live siblings, suggesting that they were initially able to grow but eventually became inviable as they grew. in most cases the viable sibling cells retained flagella, indicating the culture had not yet reached the first s phase, confirming that death occurred during g this cell death during g is consistent with increased chromosome loss during mitosis, which should result in death during growth as essential gene products become depleted. we cannot, however, rule out the possibility that the cell death results from other defects in cell division. in order to test whether supernumerary centrioles might cause the elevated cell death we observed, we asked whether the cell division defects in vfl correlated with the number of centrioles present. as previously shown, the fact that all centrioles are active as basal bodies in vfl mutants allows the number of centrioles to be inferred from the number of flagella. we therefore examined the outcome of cell divisions of cells having either the correct centriole copy number, too few centrioles or supernumerary centrioles. as indicated in fig. the fractions of inviable progeny were statistically similar for all three classes. we especially note that the cells with supernumerary centrioles did not show an increase in cell death after division. discussion these results demonstrate that the vfl2- point mutation in centrin leads to increased genomic instability via an increase in chromosome loss, suggesting that centrin plays a role in mitotic chromosome segregation. formally, these results suggest that the gene encoding centrin could act as a "housekeeper" type tumor suppressor gene, which when mutated would predispose a cell to tumorigenesis indirectly by increasing genomic instability. the mechanism by which the vfl mutation leads to increased chromosome loss is unknown. the chromosome missegregation seen in the vfl centrin mutant is reminiscent of the ipl phenotype seen in yeast mutants defective in the yeast centrin homolog cdc, which is presumably due to a failure to form a mitotic spindle. by analogy with cdc mutants, it is then possible that the chromosome loss in vfl results from defective spindles. however, unlike in yeast where the spindle pole body is absolutely necessary to form a mitotic spindle, in higher eukaryotes it has been shown that bipolar spindles can self-assemble in the absence of centrioles or centrosomes. thus it was not obvious a priori that a defect in a centriole-associated protein should have any effect on mitosis. indeed, in the specific case of the chlamydomonas vfl mutant, it has previously been reported that even though the vfl mutation causes centrioles to detach from the spindle poles, the acentriolar spindles are nonetheless still bipolar. on this basis it was concluded by wright and coworkers that centrin does not play a role in mitosis. it might therefore seem surprising that we find an increase in chromosome loss in the vfl mutant. however, while the ability of cells lacking centrioles or centrosomes to form bipolar spindles is well documented, and these spindles can be seen to undergo an apparently normal anaphase chromosome separation, the question of whether such acentriolar spindles might have an altered fidelity of chromosome segregation compared to normal spindles has never been directly addressed. we speculate that the chromosome missegregation we observe in the vfl mutant reflects a decreased effectiveness of acentriolar spindles that self-assemble in this mutant. this would suggest a mitotic fidelity function for centrioles, which requires not merely the presence of centrioles in the cell, but also their specific localization at the spindle pole. alternatively, the chromosome loss we observe could be a byproduct of multipolar spindles. a small fraction of vfl cells have three or more centrioles that could in theory give rise to tripolar or tetrapolar spindles. we suspect this is not the case because only bipolar spindles have been observed in these mutants. moreover, since centrioles are not located at spindle poles in this mutant, it is unlikely that the number of centrioles would have any effect on the number of spindle poles. the lack of centrioles at the poles also rules out configurations such as those seen during mitosis in multinucleate ptk cells, when multiple centriole pairs line up on either side of a single metaphase plate to form a masked multipolar spindle. furthermore, to the extent that production of inviable cells during mitosis reflects chromosome loss, the data of fig. argue against multipolar spindles playing a dominant role in chromosome loss since cells containing supernumerary centrioles do not show any increase in the rate of production of inviable progeny cells. it is also possible that the chromosome loss in vfl mutants arises through a spindle-independent mechanism. because centrin may be involved in nuclear transport, it is possible that the vfl mutation could cause defects in chromosome organization due to a nuclear trafficking defect, leading indirectly to chromosome loss. we also note that disruption of centrin by rnai in chlamydomonas has been reported to cause cytokinesis defects leading to binucleate cells. however such a cytokinesis defect cannot explain our results because the loh assay we employed selects for loss of act rather than the presence of additional copies. detailed cell-biological analysis of centrin mutant cells during division will be necessary to understand the basis of the chromosome loss we have observed.  we have found that a mutation in the centriole-associated protein centrin, which causes affected cells to form acentriolar spindles and lack centrin-based fibers, produces an increase in genomic instability caused by an increase in the rate of chromosome loss. these results indicate that either centrioles or the centrin protein itself are involved in increasing the fidelity of mitotic chromosome segregation. 
live cell imaging reveals an increased rate in cell death during g in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process. the role of centrioles in mitotic spindle function remains unclear. using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. the increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity. one approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability. we addressed the role of the centriole-associated ef-hand protein centrin in genomic stability using a chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus.
 the role of centrioles in mitotic spindle function remains unclear. one approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability.  we addressed the role of the centriole-associated ef-hand protein centrin in genomic stability using a chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus. using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. live cell imaging reveals an increased rate in cell death during g in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process.  the increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity. 