Mutations in the alpha-tubulin 67C gene specifically impair achiasmate segregation in Drosophila melanogaster

Abstract

Drosophila melanogaster oocytes heterozygous for mutations in the alpha-tubulin 67C gene (alphatub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the alphatub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase.

Figure 1

Bipolar spindle assembly and failure of chromatin stretching in oocytes expressing αtub67C ^P40. A, Drosophila female meiotic spindles were examined by indirect immunofluorescence using antitubulin antibodies. Shown are maximum intensity projections of oocytes heterozygous for the X balancer chromosome, FM7, with either 2 copies of wild-type αtub67C genes (top row) or with one copy of wild-type αtub67C and one copy of the αtub67C^P40mutant version of αtub67C (bottom row). Bar, 10 μm. B, FM7/X oocytes with the indicated genotypes were immunolabeled with both antitubulin and anticore histone antibodies and analyzed by confocal microscopy. Shown are maximum intensity projections of the chromatin masses of oocyte meiotic spindles. Chromatin masses from wild-type oocytes are shown in the top row and, in the next two rows, masses from oocytes heterozygous for the αtub67C^P40mutation are shown. Chromatin masses from oocytes heterozygous for both nod and αtub67C^P40 are shown in the bottom row. Bar, 4 μm. Oocytes were prepared and examined as previously described with minor modifications (Theurkauf and Hawley 1992; Matthies et al. 1996).

Figure 2

Quantitation of the failure of chromatin stretching due to the αtub67C ^P40 mutation. Shown are plots of chromatin length distributions in the indicated genotypes (A, B, and C), spindle versus chromatin length (D, E, and F), and finally, spindle length versus axial ratio of the chromatin (chromatin length/chromatin width) (G, H, and I).

Figure 3

Failure of centromere positioning (MEI-S332 protein) in heterozygous αtub67C ^P40 mutant oocytes. FM7/X oocytes with the indicated genotypes were immunolabeled with anticore histone, antitubulin, and anti–MEI-S332 antibodies. The first column displays the projections of MEI-S332 alone (red, whereas the second columns displays the projections for both histone [green] and MEI-S332 [red]). In the first column are the projections of the MEI-S332 data, and the second column is the merged projection of the histone and MEI-S332 data. The top two rows depict oocytes heterozygous for αtub67C^P40. In the oocyte displayed in the top row, a portion of the MEI-S332 immunoreactivity is found adjacent to the main chromosome mass, whereas the remainder of the protein is detected toward the center of the chromosomal mass. In the second panel, virtually all of the MEI-S332 immunoreactivity remains at one pole. The middle two rows are oocytes heterozygous for both αtub67C^P40and nod (nod^b17). In each of these oocytes, the MEI-S332 immunoreactivity is seen at opposite poles of the lengthening chromosomal mass, as is observed in wild-type oocytes (bottom row). Bar, 4 μm.

Figure 4

The αtub67C^P40mutation leads to elevated levels of achiasmate chromosome missegregation. The frequencies of X (dark bars) and 4^th (light bars) chromosome missegregation (nondisjunction) are displayed for various genotypes. The genotype at the αtub67C locus is represented as: +/+, two wild-type copies of the αtub67C gene; +/P40, heterozygous for the αtub67C^P40 mutation; and +/P40Δ, heterozygous for the αtub67C^P40 mutation. These experiments were done in both X/X and X/FM7 females, allowing us to examine segregation in oocytes with (+) or without (−) exchange on the X chromosome, respectively. N is the adjusted total of progeny scored (Hawley et al. 1993). Chromosome segregation was monitored by methods outlined in Hawley et al. 1993 and Sekelsky et al. 1999. Although females homozygous for αtub67C^P40are sterile, αtub67C^P40Δ/+ females are fully fertile and viable.

Figure 5

The fidelity of achiasmate chromosome segregation is sensitive to the dose of both Nod and αtub67C proteins. Dark and light bars represent the frequencies of X and 4^th chromosome missegregation, respectively, in the indicated genotypes. A, The effects of heterozygosity for αtub67C mutations on chromosome missegregation can be suppressed by heterozygosity for a loss-of-function allele of nod (nod^b17). B, Increasing the dose of nod ⁺ to three copies (by use of a duplication) increases the frequency of X and 4^th chromosomal missegregation, both in the females carrying two normal copies of the αtub67C gene, and, to an even greater extent, in females heterozygous for a mutation in the αtub67C gene. This enhancement of missegregation by the nod ⁺ duplication can be eliminated by using females that simultaneously bear a nod mutation on one of the two X chromosomes (i.e., flies that only carry two copies of nod ⁺, despite carrying the nod ⁺ duplication; data not shown). This demonstrates that the enhancement observed in the presence of the duplication is due to the extra copy of nod ⁺, since both X and 4^th chromosome nondisjunction occur with the same frequencies in flies carrying two alleles of nod ⁺, regardless of whether one comes from a duplication or from both the endogenous genes on the same chromosome. C, Comparison of the effects of one, two, and three doses of nod ⁺ in FM7/X; +/P40 females. N is the adjusted total of progeny scored (Hawley et al. 1993).