The epsilon-subunit of mitochondrial ATP synthase is required for normal spindle orientation during the Drosophila embryonic divisions

Abstract

We describe the maternal-effect and zygotic phenotypes of null mutations in the Drosophila gene for the epsilon-subunit of mitochondrial ATP synthase, stunted (sun). Loss of zygotic sun expression leads to a dramatic delay in the growth rate of first instar larvae and ultimately death. Embryos lacking maternally supplied sun (sun embryos) have a sixfold reduction in ATP synthase activity. Cellular analysis of sun embryos shows defects only after the nuclei have migrated to the cortex. During the cortical divisions the actin-based metaphase and cellularization furrows do not form properly, and the nuclei show abnormal spacing and division failures. The most striking abnormality is that nuclei and spindles form lines and clusters, instead of adopting a regular spacing. This is reflected in a failure to properly position neighboring nonsister centrosomes during the telophase-to-interphase transition of the cortical divisions. Our study is consistent with a role for Sun in mitochondrial ATP synthesis and suggests that reduced ATP levels selectively affect molecular motors. As Sun has been identified as the ligand for the Methuselah receptor that regulates aging, Sun may function both within and outside mitochondria.

Figure 1.—

Organization of the sun genomic locus. The genomic region around the sun locus deduced from restriction mapping, partial sequencing, and genome project data is shown. Identified genes are shown on the chromosome, with the orientation of the gene shown by the lines linking exons above the line (5′ to 3′ is from left to right) or below the line (5′ to 3′ is from right to left). The 11-kb SalI genomic rescue fragment is shown as a bar below the chromosome.

Figure 2.—

Sequence of the sun gene. (A) DNA sequence of the sun gene, with the predicted amino acid sequence of the gene product shown at bottom. The mutation found in sun alleles 1–3 that changes Trp4 to a stop codon is highlighted with asterisks above the DNA sequence. Vertical bars indicate the location of splice sites; the first intron is 129 bp long, and the second is ∼600 bp. The unspliced transcript is predicted to be ∼1.3 kb. (B) Alignment of the Sun amino acid sequence with the ε-subunits of ATP synthase from a representative range of organisms. Residues common to three or more sequences are boxed. The amino acid sequence accession numbers are: Drosophila CG31477, NP_731449; Arabidopsis, Q96253; C. elegans, P34539; Human, NP_008817; S. cerevisiae, NP_015052; and Schizosaccharomyces pombe, NP_596577. The C. elegans and S. pombe sequences are putative proteins identified by analysis of genome projects data.

Figure 3.—

ATPase activity in wild-type and sun mutant embryo extracts. Bars indicate ATPase activity in high-speed pellets made from embryos collected from wild-type and sun³/+ stocks or germline mosaic females of sun³ and sun¹. Optical density (A₆₆₀) values and genotypes are indicated along the ordinate and abscissa, respectively. The total ATPase activity derived from the mitochondrial ATP synthase is indicated by the purple portion of each bar. ATPase activity was measured in the absence (purple plus white) and presence (white) of the mitochondrial ATP synthase-specific inhibitor NaN₃ (Bowman et al. 1978). Each protein fraction was assayed twice and the average values are shown.

Figure 4.—

Nuclear behavior of wild-type and stunted embryos. Wild-type (left column) and sun^mat− embryos (stunted, right column) were stained with the nuclear dye propidium iodide. During the early cortical divisions, slight abnormalities are observed in sun^mat− embryos. During the late cortical divisions, areas in which large numbers of nuclei have fallen back into the center of the embryo are observed and spacing between nuclei has become irregular. At telophase of cycle 13, the spacing defects are readily apparent as nuclei come closer together than in wild type, forming lines of nuclei arranged in clumps in a manner reminiscent of the “paisley” pattern. The 60× insets reveal many nuclei are in contact with their neighbors and form lines of touching nuclei. At cellularization, nuclear fusion has occurred throughout the sun^mat− embryos; this is apparent in the 60× inset. Bar, 10 μm.

Figure 5.—

Nuclear dynamics in a sun^mat− embryo. Images of living sun^mat embryos injected with fluorescently labeled histones at nuclear cycle 13 are shown. Prophase (a), metaphase (b), anaphase (c), telophase (d and e), and interphase (f) are shown. Fusions between dividing nuclei occur at telophase (see arrows in e).

Figure 6.—

Actin distribution in sun^mat− embryos. Wild-type (sun^mat−/FM7c) and sun^mat− (stunted) embryos were stained with fluroscein-labeled phalloidin and propidium iodide. The actin caps appear to form normally in sun^mat− (stunted) embryos (A and B). However, the metaphase furrows are frequently absent in sun^mat− embryos (D). By cellularization, the normal orderly outline of cells (E) is severely disrupted (F). Bar, 10 μm.

Figure 7.—

Analysis of microtubule behavior in a sun^mat− embryo. (A) Fixed analysis of wild-type vs. sun^mat− embryos. (B) Time-lapse sequence of a sun^mat− syncytial blastoderm embryo injected with fluorescently labeled tubulin. (a and b) Prophase. The syncytial nuclei can be seen by an absence of tubulin staining due to exclusion by the nuclear envelope. The position of the centrosomes can be determined from increased density of tubulin on either side of the nuclei. On the left, a line of nuclei (indicated by arrows) in which the centrosomes have aligned can be seen. (c) During metaphase, the mitotic spindles corresponding to the aligned centrosomes can be seen on the left. (d) During late metaphase, irregular mitotic figures become apparent, with inappropriate interactions between neighboring spindles, notably at the bottom left and right corners. (e) Anaphase. A lack of mitotic coordination becomes apparent as some spindles break down more quickly than others, and midbodies form (accumulation of microtubules at the spindle equator; these are more apparent in f). (f) Telophase. The position of the centrosomes can be deduced from small local concentrations of tubulin; midbody formation has occurred at the remaining spindles. A group of tightly apposed abnormal midbodies can be seen in the bottom left (indicated by arrows).