Influence of centriole number on mitotic spindle length and symmetry

Abstract

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

Figure 1

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.

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 abnormalities in bipolar spindles. Because categories are not all mutually exclusive, percentages can add up to greater than 100.

Figure 3

Quantifying chromosome congression in centriole mutants. (A) Area. (B) Shape Factor, as defined in text.

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 that the normal number of centrioles in a wild-type cell at metaphase is 4, two at each pole. A total of 125 spindles from vfl1, vfl2, and vfl3 mutants were combined to form this histogram. The following number of total spindles was counted for each centriole number class (0, n=5; 1, n=8; 2, n=20; 3, n=19; 4, n=39; 5, n=13; 6, n=12; 7, n=9).

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 length versus the number of centrioles found at a pole. A total of 184 bipolar spindles were analyzed from vfl mutants for each plot. Error bars are standard error of the mean.

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 length, plotted versus the absolute value of the difference in number of centrioles between the two poles. (B) Asymmetry in pole width, defined as the ratio of the width of the larger pole to the smaller pole, plotted versus the absolute value of the difference in number of centrioles between the two poles. (C) Asymmetry in half-spindle tubulin content, defined as the ratio of the background-corrected integrated tubulin intensity in the two half-spindles, plotted versus the absolute value of the difference in number of centrioles between the two poles. A total of 184 bipolar spindles from vfl mutants were tabulated for these plots. Error bars are standard error of the mean.

Figure 7

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).

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 (n=69, 60,160, 93, and 17, respectively, for cells with 0,1,2,3, and 4 centrioles).

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 of cell divisions in which one or both daughter cells were dead analyzed in vfl2 mutants and plotted versus the number of centrioles in the mother cell.