The Drosophila meiotic mutant mei-352 is an allele of klp3A and reveals a role for a kinesin-like protein in crossover distribution

Abstract

The semisterile meiotic mutant mei-352 alters the distribution of meiotic exchanges without greatly affecting their total frequency. We show that the mei-352 mutation is an allele of the klp3A gene, which encodes a kinesin-like protein of the Kinesin-4 family. The semisterility observed in mei-352 females results from a known defect of klp3A oocytes in mediating pronuclear fusion. Interestingly, other klp3A alleles also exhibit defects in meiotic recombination similar to those of mei-352. Finally, we show that the Klp3A protein localizes within the oocyte nucleus during meiotic prophase, the time at which exchange distribution is established, and extensively colocalizes with DNA. The parallel of the klp3A phenotype with a meiotic defect observed for kar3 mutants in yeast suggests a role for kinesins in early meiosis and might reflect a previously suggested role for this class of kinesins in chromosome condensation.

Figure 1.—

Alterations of meiotic exchange distribution are a general feature of klp3A mutants. Genetic map distances relative to wild type were calculated by dividing the map distance by the map distance for the Df/+ for the same interval. These data are plotted on the y-axis for four intervals on the left arm of chromosome 2 (x-axis). Df, Df(1)ED6579.

Figure 2.—

Schematic of the 1212-amino-acid Klp3A protein, showing domain structure and locations of identified mutations. Klp3A contains an N-terminal kinesin-like motor domain (aa 1–342, hatched area), a coiled-coil-rich stalk domain (aa 343–996, open area), and a C-terminal tail domain (aa 997–1212, stippled area). The locations and nature of four point mutations in klp3A identified in this study (klp3A⁵²¹, klp3A^mei-352, klp3A⁸³⁵, and klp3A¹¹²⁴) are indicated above the protein.

Figure 3.—

Phenotype of embryos from klp3A females. In A–E, embryos are stained with antihistones (green) to visualize chromatin and antitubulin (red) to visualize microtubules. (A) Embryo from a klp3A^mei-352/FM7w female in mitotic cycle 2. Two zygotic nuclei are present on mitotic spindles with centrosomes at each pole (small arrows). The polar body chromatin mass is visible at left (large arrow). (B) Low-magnification image of an embryo from a klp3A^mei-352 homozygous female showing that only two masses of chromatin are present: a large mass of polar body chromatin and a smaller nucleus on a spindle. (C) Higher-magnification image of the embryo from B. The mass of polar body chromatin is associated with a circular concentration of microtubules (large arrow) and the small nucleus sits on a spindle (small arrow). However, the spindle appears to lack asters associated with the spindle poles but several free-lying centrosomes are present (arrowheads). (D) Embryo from a klp3A^mei-352/klp3A⁵²¹ female. The only antihistone signals are the polar body chromatin mass (large arrow) and a small nucleus residing on a spindle (small arrow). Centrosomes (arrowheads) appear to have dissociated from the spindle and additional centrosomes are visible in the embryo. (E) Embryo from a klp3A^mei-352/klp3A¹¹²⁴ female. Five independent centrosomes are visible (arrowheads), but none appear associated with the single spindle (small arrow) or polar body chromatin mass (large arrow).

Figure 4.—

Localization of Klp3A protein during early meiotic prophase. (A–D) Two optical sections from the same w¹¹¹⁸ germarium stained with anti-Klp3A (red) and anti-C(3)G (green). The germarium is oriented with the anterior tip at the top. As developing cysts (dotted outlines) move toward the posterior, the initiation of meiosis is marked by the formation of the SC, of which C(3)G is a component. Mitotically dividing germline cells display a variable localization of Klp3A, including concentration at the spindle midbody in telophase (arrowhead in B), similar to Klp3A localization in embryos (Kwon et al. 2004). Klp3A level is low in zygotene cysts, in which the SC is just beginning to form and the anti-C(3)G signal is weak and punctate (small arrows in B and D). Two pachytene 16-cell cysts that show full accumulation of C(3)G protein (large arrows in B and D) also show distinct, strong nuclear localization of Klp3A. The level of Klp3A apparently decreases in older 16-cell cysts as prophase continues, although Klp3A can be observed in the follicle cells surrounding these cysts. Bar, 10 μm. (E–H) A cell from a 16-cell cyst stained with anti-Klp3A (red, E), DAPI (blue, F), and anti-C(3)G (green, G). A merged image is shown in H. The anti-Klp3A signal shows extensive colocalization with DAPI-stained DNA and C(3)G, suggesting that the Klp3A protein may be associated with chromatin. Bar, 1 μm. (I–L) A cell from a 16-cell cyst stained with anti-Klp3A (red, I), DAPI (blue, J), and antilamin (green, K). A merged image is shown in L. The strong anti-Klp3A signal is contained within the nuclear envelope, as detected by antilamin, whereas Klp3A protein in the cytoplasm is not detected above background levels. Bar, 1 μm.