Mitotic spindle aberrations in vfl mutants
If centrioles play a role in organizing the mitotic spindle, then mutants with abnormal centriole number should show defects in spindle organization. Centriole number can in principle be altered by physical manipulation such as microdissection, cell fusion, and laser ablation, but such approaches run the risk of unintended side-effects and are hard to apply to large numbers of cells. We therefore turned to a genetic approach using the unicellular green alga Chlamydomonas reinhardtii
. A class of Chlamydomonas
mutants has been described in which centriole ultrastructure is abnormal. These include bld2
, in which centrioles are flat discs of singlet microtubules [Goodenough and St. Clair, 1975
; Dutcher et al., 2001]; uni3
in which centrioles have doublet rather than triplet microtubules [Dutcher et al., 1998
]; and bld10
in which centrioles are largely absent [Matsuura et al., 2004
]. Analysis of spindle organization in such mutants allows us to test whether centriole structure affects mitosis. A second class of Chlamydomonas
centriole-related mutants consists of the VFL mutants vfl1
, and vfl3,
in which centriole number is variable from cell to cell [Adams et al., 1985
; Wright et al., 1989
; Kuchka and Jarvik, 1982
]. In these mutants, centriole ultrastructure is apparently normal, but some cells lack centrioles, or have just one, others have supernumerary centrioles, while others have the normal wild-type number and serve as an internal control for centriole-independent effects of the mutations. These mutants allow us to explore the effect of centriole number on spindle morphology.
Using these two classes of mutants, we examined the three-dimensional organization of mitotic spindles by fluorescence microscopy. Mitotic cells were identified during initial scanning using the anti-phospho-histone antibody staining. Among the mitotic cells, metaphase cells were identified according to the description by Doonan and Grief 
who defined metaphase as the stage in which spindle poles are well separated, chromosomes are condensed, and microtubules of the spindle run all the way through the nucleus. A similar convention is followed by Ehler et al. 
who diagram prophase as a stage with condensed chromosomes but microtubules not fully extending through the nucleus. All of our analyses were conducted using spindles at this stage. We found that centriole mutants in Chlamydomonas
displayed a range of abnormal spindle phenotypes, as illustrated in . Defects in spindle morphology included monopolar spindles, multipolar spindles, split or multiradial spindles, and asymmetric bipolar spindles with unequal pole focus. These defects occur rarely, if ever, in wild-type cells, but were seen in all mutants examined. Similar defects also occur in asq2
mutant cells, which have defects in mother-daughter centriole cohesion [Feldman and Marshall, 2009
]. We frequently observed centrioles in these mutant cells which were not associated with any spindle pole. These detached centrioles, defined as those further than 1 μm from a pole, still appeared able to induce microtubule organizing centers (MTOCs) as judged by the appearance of small asters around each detached centriole during metaphase. We have previously observed that centrioles unassociated with spindles are able to form MTOCs (Feldman and Marshall, 2009
). We never observed clustering of extraneous centrioles such as has been reported in some mammalian cell lines [Quintyne et al., 2005
Spindle phenotypes seen in mutants with abnormal centriole number or ultrastructure. (red) anti-POC1 to visualize centrioles. (green) anti-alpha-tubulin. (blue) anti-phospho-histone H3.
Comparing different types of mutants, we find that a similar spectrum of defects was seen in mutants with abnormal centriole ultrastructure and in mutants with abnormal centriole number distributions (). The frequency of monopolar spindles was significantly increased for all mutants when compared with wild type (P<0.05 by Fisher’s exact test corrected for multiple comparison). In contrast, the frequency of multipolar spindles was not statistically different from wild-type in any mutant except for vfl1, in which the difference was significant (P<0.02 by Fisher’s exact test). This result suggests that formation of multipolar spindles is a relatively rare event in these mutants, whereas formation of monopolar spindles is far more common. Overall, however, the majority of cells were still able to form bipolar spindles in all of the mutants examined (), suggesting that the robustness of bipolar spindle self-assembly can generally overcome the influence of centriole number variation.
Figure 2 Quantifying spindle defect frequency in individual mutants. Graph shows fraction of mitotic spindles in each genetic background (signified by color of the bars) that show each of the listed structural characteristics. (A) Spindle polarity. (B) Morphological (more ...)
Even when bipolar spindles formed, in many cases these showed clear abnormalities in appearance (). As shown in , the frequency of spindles with unequal distributions of tubulin in the two half spindles (as judged by eye - see below for quantification of asymmetry) was significantly increased in the set of mutants (P<0.05 for vfl1, vfl3, and bld2 individually, P<0.007 for the group of mutants collectively), as was the frequency of spindles with unequal pole focus (P<0.05 for all mutants individually, P<0.0006 for the whole set of mutants collectively), and the frequency of detachment of centrioles from the poles (P<0.05 for all mutants individually, P<0.0001 for the set of mutants collectively). The high frequency of detachment suggests the effect of supernumerary centrioles may be to some extent buffered by the inability of all centrioles to become associated with spindle poles.
Defects in spindle morphology may lead to defects in chromosome segregation, but because we rely on imaging of fixed cells to score spindle morphology, we cannot follow the subsequent chromosome segregation mediated by such spindles. However, as another function of the mitotic spindle is to mediate congression of chromosomes to the metaphase plate [Khodjakov et al., 1999
], we asked whether the defective spindles that form in centriole mutants might also be defective in congression. We scored the compactness of the metaphase plate by measuring the area of the phospho-histone specific staining in two-dimensional image planes in which both poles are in focus, with results graphed in . By this measure, several centriole mutants show a small but statistically significant increase in chromatin area (P<0.0001 among the group by ANOVA, P<0.05 for vfl1
, and bld10
considered individually by Holm-Sidak test). These three mutants that showed alteration in chromatin area also showed inequality in tubulin between the half spindles (). We note that the area analyzed here is not the area of individual chromosomes, but the area subtended by the entire metaphase plate. In addition to area, a "shape factor" was calculated by taking the ratio of the circumference of the entire chromatin mass to the square root of the area (). This is a unit-less quantity that takes its minimum value if the metaphase plate region has a circular cross-section, and has a larger value if the chromosome-containing region becomes more elongated. The alteration in chromatin mass shape factor was significantly different from wild type in vfl1
, and bld10
mutants (P<0.05 in all cases individually by Holm-Sidak test and P<0.0001 among the entire group of mutants considered versus wild type by ANOVA), which we interpret as an average elongation of the metaphase chromosome cluster along the axis of the spindle, as though the chromosomes had not fully congressed to the center. These defects in metaphase plate size and shape may suggest that the alterations in spindle morphology seen in these mutants result in subtle defects in chromosome congression, although our data cannot rule out an effect on chromatin compaction.
Quantifying chromosome congression in centriole mutants. (A) Area. (B) Shape Factor, as defined in text.
Spindle polarity versus centriole number
The results of the previous section indicate that vfl mutants in Chlamydomonas, which have variable numbers of centrioles per cell, can form both monopolar and multipolar spindles. These defects might reflect a direct role for centrioles in spindle assembly, but two alternative explanations exist - the defects might reflect aberrations in cellular organization caused in earlier divisions, or the defects might reflect additional, centriole-independent functions of the VFL genes. To distinguish these possibilities, we scored the frequency of monopolar, bipolar, and multipolar spindles as a function of centriole number (). For this plot we grouped all three vfl mutants, vfl1, vfl2, and vfl3, together on the grounds that they have indistinguishable distributions of centriole number. This result shows that cells with fewer than four centrioles at the time of mitosis are more likely to form monopolar spindles, while cells with more than four centrioles are more likely to form multipolar spindles. However, in some cases cells with less than four centrioles still formed multipolar spindles, and cells with more than four centrioles still formed monopolar spindles.
Figure 4 Spindle polarity versus centriole number in metaphase spindles of vfl mutants. Histogram shows the fraction of mitotic cells with either monopolar (blue) or multipolar (red) spindles, as a function of the total number of centrioles in each cell. Note (more ...)
The frequency of bipolar spindles was maximum for cells containing exactly four centrioles. The fact that polarity correlated with number of centrioles is not consistent with a centriole-independent side effect of the vfl mutations, because the cells analyzed shared the same pool of genetic defects and differed only in centriole number.
Spindle geometry versus local centriole number at the pole
If centrioles exert a direct influence on spindle assembly, then we might expect to see a correlation between the centriole number at a given pole and the morphology of the half-spindle containing that pole. Because the primary proposed function for centrioles at the poles is to recruit a functional MTOC and thus contribute microtubules for spindle assembly, we specifically tested whether increased centriole number would correlate with either the width of the pole or the length of the half-spindle. If centrioles help recruit microtubule nucleating activity at the poles, one would expect that more centrioles would lead to more microtubules and thus a wider or thicker half-spindle. On the other hand, if centrosomes play a key role in pulling spindle microtubules into a tight focus [Silk et al., 2009
], then more centrioles at a pole might lead to a narrower pole. In order to explore these possibilities, we describe half-spindle morphology by two parameters, the width of the pole and the length of the half-spindle, as illustrated in , and ask if either parameter varies as a function of centriole number associated with each pole. We define the pole-associated centriole number as the number of centrioles that are within one micron of the nearest pole.
Figure 5 Geometric features of bipolar spindles correlate with centriole number at poles. (A) diagram illustrating the two parameters measured, pole width and half-spindle length. (B) Spindle pole width versus the number of centrioles found at a pole. (C) Half-spindle (more ...)
A clear trend was seen relating centriole number at a pole to the width of the pole (), such that when the number of centrioles at a pole exceeded 2, the width of the poles became significantly increased (average width 0.53 μm ± 0.03 μm SEM versus 0.75 μm ± 0.09 μm SEM, P<0.02). For numbers between 0 and 2, there was no difference in the average width, indicating that the presence of too few centrioles at a pole, or even none at all, has little or no effect on pole focusing. Overall the correlation coefficient between the number of centrioles at a pole and the width of the pole was 0.92. The ability of poles to focus in the absence of centrioles, even in a cell type normally containing centrioles at the poles, is consistent with previous studies (Manning and Compton, 2007
A clear trend was also seen relating the centriole number at a pole to the length of the corresponding half-spindle (). When examining this relation, we found no variation in the average spindle half length for centriole numbers between 1 and 3, but found that poles lacking centrioles had a somewhat increased length for the corresponding half spindle (average half length 2.16 μm ± 0.09 μm SEM for zero centrioles versus 1.99 μm ± 0.04 μm SEM for one to three , P<0.05), while poles with 4 or more centrioles had a decreased average half-spindle length (average half length 1.71μm ± 0.15 μm SEM for four or more centrioles versus 1.99 μm ± 0.04 μm SEM for one to three , P<0.07). The difference in half length corresponding to poles with too few centrioles and poles with too many centrioles was highly significant (P<0.02 for zero centrioles versus four or more centrioles). Overall the correlation coefficient was −0.92 for spindle half length versus the number of centrioles at a pole. In contrast to these clear trends relating centriole number to half-spindle length and pole width, we found no evidence for a correlation between total tubulin content of each half spindle, as judged by background-corrected integrated tubulin immunofluorescence intensity, and the number of centrioles as the pole (), for which we calculated a correlation coefficient of 0.18. This negative result does not lead to a strong conclusion because quantification of protein content based on immunofluorescence is problematic, due to the inherent nonlinearity of antibody-epitope interactions as well as high variability in fixation and antibody penetration between samples. It is therefore quite possible that this high variability might mask an actual trend, if present.
The results of suggest a link between centriole number at a pole and the local geometric properties of the spindle at that pole, but they ignore interactions between the two half spindles. We therefore asked whether inequality in the number of centrioles at the two poles of a spindle might lead to increased asymmetry between the two halves of the spindle. To do so, we plotted the average ratio between the lengths of the longer and shorter half spindles (a measure of asymmetry in spindle length) versus the absolute difference in centriole number between the two poles of each spindle (). The graph indicates that length asymmetry as defined here increases monotonically with increasing centriole imbalance, with an overall correlation coefficient of 0.96 between length asymmetry and centriole imbalance. This result indicates that increased imbalance in centriole number does in fact correspond with increased asymmetry in the spindle half lengths. In contrast, we see no such relation between imbalance in centriole number and asymmetry of spindle pole widths (), for which we calculate a correlation coefficient of only 0.31.
Figure 6 Imbalance in centriole number between two poles of a bipolar spindle correlates with asymmetry in half-spindle lengths but not pole width. (A) Asymmetry in half-spindle length, defined as the ratio of the larger half-spindle length to the smaller half-spindle (more ...)
Likewise, we failed to detect any significant correlation between centriole number imbalance and asymmetry in tubulin content as judged by immunofluorescence intensities (). We found a correlation coefficient of only 0.15 for tubulin asymmetry versus centriole number difference. As discussed above, quantification of tubulin by immunofluorescence is inherently problematic, so no strong conclusion can be drawn from this negative result. We note also that the variation of half-spindle geometry with centriole number does not necessarily require a variation in tubulin quantity, since as centriole number increases, half-spindle length decreases but pole width increases. If we imagine that each half spindle is shaped like a truncated cone, we see that increasing the width will tend to increase the volume of the cone, while decreasing the length will tend to decrease the volume. The combined effect of a width increase and a length decrease will therefore only lead to a slight overall variation in the volume of the half spindle, which, combined with the difficulties of quantifying protein content from immunofluorescence intensity, would be unlikely to be detected in our experiments.
Cytokinesis defects in vfl mutants
In addition to a putative role in mitosis, centrioles have also been implicated in regulating cytokinesis, either by signaling to the cytokinesis machinery itself [Gromley et al., 2003
; Piel et al., 2001
; Murata-Hori and Wang, 2002
] or by coordinating the orientation and position of the spindle with the cleavage furrow [Costello 1961
; Ehler et al., 1995
; Hinchcliffe et al., 2001
; Von Dassow et al., 2009
]. As with mitosis, it is well established that cleavage can progress without centrioles and even without spindles at all [Hiramoto 1965
], so if centrioles contribute to the process at all, they would only be expected to act in a regulatory manner. Defects in cytokinesis have been observed in some instances after centriole ablation [Khodjakov and Rieder, 2001
], but only in a fraction of cells. Therefore, cells with structural or numerical centriole abnormalities might only show a small increase in cleavage defect frequency over normal cells, requiring a statistical test on a large number of cells, an approach facilitated by genetic the genetic mutations available in Chlamydomonas
To test for an effect of abnormal centriole number on cytokinesis we apply the same strategy as above, using mutants with variable centriole number to ask whether cytokinesis defects occur at a significantly increased rate when centriole number is abnormal. Observations of populations of vfl mutant cells show frequent failures of cytokinesis, especially incomplete cleavage furrows (). To quantify cytokinesis failure in vfl mutants, we embedded single vfl mutant cells and observed them before and after cell division. As tabulated in , we see a dramatic increase in the frequency of cleavage failure in vfl mutants as compared with wild-type cells. The increase in frequency is statistically significant (P<0.05 for both vfl2 and vfl3 compared to wild-type using Fisher's exact test corrected for multiple comparison). For vfl1 mutants, there was no statistically significant increase in cytokinesis defects compared to wild type. Given that in all other respects, such as centriole number distribution, vfl1 and vfl3 seem to have identical phenotypes, we were surprised to see this difference and we currently do not know why this difference exists.
Defects in cytokinesis in mutants with abnormal centriole number. (A) images of normal and defective cleavage. (B) Frequency of cytokinesis defects in mutants (wt n=103; vfl1 n=129; vfl2 n=401; vfl3 n=60).
These results indicate that variable centriole number mutants in Chlamydomonas show a significant increase in cytokinesis defects. These defects might reflect a direct role for centrioles in cytokinesis, but as with the mitotic analysis described above, two alternative explanations exist - the defect might reflect aberrations in cellular organization caused in earlier divisions, or the defects might reflect additional, centriole independent functions of the VFL genes.
One approach for distinguishing direct effects of centriole number from centriole independent side-effects of the mutations is to use a conditional vfl2
allele [Taillon, 1993
]. We grew vfl2ts
mutants for 1 week at the nonpermissive temperature, shifted them back to the permissive temperature for 2 days, and then embedded and tracked individual cells in agarose pads. We have previously demonstrated [Marshall, 2007
] that under this growth regime the vfl2ts
mutant strain shows a complete restoration of centrin fiber assembly and centriole segregation within 1 day of growth after returning the strains to the permissive temperature, suggesting that by the time we started observing cells, the molecular function of centrin was restored. However, we have also found the number of centrioles per cell takes additional generations to become fully corrected [Marshall, 2007
]. If such cells showed normal cleavage despite having variable numbers of centrioles, it would indicate that the cleavage failures observed in constitutive vfl2
mutants must have been caused by a centriole-independent effect of the vfl2
mutation. However, as shown in , we found that in vfl2ts
mutants shifted back to the permissive temperature and grown for over 1 day, the defects in cytokinesis continue to occur at a frequency that was not significantly different from the constitutive vfl2
mutant. This result suggests the defects are caused by the abnormal centriole number and not the loss of centrin function, since by this time the latter had been restored following the downshift to permissive temperature, as judged by centrin fiber assembly.
Figure 8 Correlating cytokinesis defects with centriole number abnormality. (A) Defects following restoration of normal VFL2 gene function following down-shift of a conditional mutant (vfl2ts n=12). (B) defect frequency versus centriole number in vfl2 mutants (more ...)
A second approach to testing whether the cytokinesis defects are centriole-dependent is to ask whether cytokinesis defects correlate with centriole number. We tested this correlation by embedding single vfl2
cells in agarose pads and observing them before and after division, making note of the number of flagella in the parent cell prior to division. Since in vfl2
all centrioles are active as basal bodies, the number of flagella can be taken as a reliable indicator of the number of centrioles, as previously demonstrated using multiple protein markers as well as electron microscopy [Marshall et al., 2001
]. We then asked if cytokinesis defect rates correlate with flagellar number, and hence centriole number. If the cytokinesis failure was caused by a centriole-independent effect of the vfl2
mutation, the failure should occur in all cells at a rate independent of the number of centrioles that happen to be present. As seen in , the rate of cleavage failure appears to vary only weakly with the number of centrioles, and does not reflect a statistically significant difference (χ 2=6.66)
Taken together these results suggest that the cytokinesis defects may have been caused by centriole number abnormalities in previous cell divisions, because the defect is still seen when the conditional vfl2 mutant is shifted back to permissive temperature, but does not correlate strongly with the number of centrioles present in the cell at the time of division.
Cell death in centriole mutants
Consistent with the increased frequency of abnormalities in mitosis and cytokinesis described above, we have observed a significantly increased proportion of dead cells in cultures of all centriole mutants relative to wild-type cells (, P<0.0001 for difference between groups, P<0.05 for each mutant individually compared with wild type), although from these data alone it is not possible to determine the proximal cause of death. Presence of increased cell debris has previously been noted for vfl3
mutants [Wright et al., 1983
]. An accumulation of dead cells could be due to a decreased rate of cell degradation or clearance from the culture, rather than an increase in death rate. To distinguish these possibilities we observed individual mutant cells embedded in agarose and measured the frequency with which a normal-looking cytokinesis gives rise to dead progeny cells. This analysis () showed increased cell death rate in mutants with abnormal centriole numbers.
Figure 9 Cell death in centriole mutants. (A) Percent of dead cells in centriole mutant populations as assayed by erythrosine staining. (B) Frequency of cell divisions in which one or both daughter cells were dead, plotted for different mutants. (C) Frequency (more ...)
As with the spindle and cytokinesis defects discussed above, the increase in cell death could either be due to the centriole abnormalities or to a centriole-independent side effect of the mutations. We therefore correlated the frequency of cell death with the number of centrioles present in vfl2
cells, using the number of flagella as an indicator of centriole number, as was done above for cytokinesis defects. The results of this analysis () indicate that the frequency with which a normal cleavage division produces one or more dead progeny is a function of centriole number, with the frequency of cell death much higher for cells with one or four centrioles compared to cells with two (P<0.02, P<0.01, respectively according to Fisher's exact test). Our results do not allow us to distinguish cell death that results from mitotic failures from death due to other causes such as increased stress sensitivity that has been reported in cells lacking centrosomes [Uetake et al., 2007