Microtubule-depolymerizing kinesin KLP10A restricts the length of the acentrosomal meiotic spindle in Drosophila females

Abstract

During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation.

Figure 1

KLP10A localization in the female meiotic and embryonic mitotic spindles. Spindles from late-stage oocytes fixed with formaldehyde/heptane (A and B) and syncytial-stage embryos fixed in methanol (C and D) were examined for the localization of endogenous KLP10A (A and C) and HA-tagged KLP10A (B and D). The HA-tagged KLP10A (B and D) was expressed in a wild-type background. (A) In oocytes, endogenous KLP10A localized throughout the meiotic spindle. (B) HA-tagged KLP10A localized throughout the meiotic spindle, but was heavily concentrated at spindle poles. In addition, the “curly pole” phenotype caused by expression of the transgene is observable (see Figure S1). (C and D) In embryos, KLP10A primarily concentrates toward the spindle poles. Microtubules were not imaged in C. In all images, DNA is shown in blue and microtubules are shown in green. KLP10A is in red in merged images (A–D) and in white in single channel images (A′–D′). Bars, 5 μm.

Figure 2

Generation and characterization of Klp10A germline mutants. (A) Klp10A coding sequence is shown with boxes representing exons. The UTRs are not shown. The hatched box indicates the region encoding the portion of KLP10A used to raise the anti-KLP10A antibody (Rogers et al. 2004). The black box indicates the region encoding the motor domain of KLP10A. The P-element (EY09320) used to generate deletions of Klp10A coding sequence is depicted by a black triangle. The sequence deleted by the Klp10A²⁴ allele is shown below with brackets surrounding the deleted region. (B) Western blot showing KLP10A expression in late-stage oocytes. Endogenous expression of full-length KLP10A is eliminated in Klp10A²⁴ germline clones (lane 2) and severely knocked down in Klp10A RNAi (lane 3) compared to wild type (lane 1). HA-tagged KLP10A is expressed at levels comparable to endogenous KLP10A (lane 4). Tubulin serves as a loading control in all lanes.

Figure 3

Microtubule and DNA disorganization in Klp10A germline mutant embryos. Embryos produced by Klp10A germline mutants show severely disorganized DNA and microtubule structures. Chromosomes are dispersed throughout the cytoplasm, and microtubules form large asters surrounding the dispersed chromosomes. See Figure 1 for wild-type embryo spindles. Also shown are two examples of embryos lacking Subito (by RNAi, see Materials and Methods). About half of the embryos show only the female polar body (arrow) and the male pronucleus (inset). Drosophila female meiosis does not segregate chromosomes into a separate polar body. In the other half of the embryos, there are nuclei attempting to divide, which may have originated from the haploid male genome. DNA is in blue and microtubules are in green. Bars, 10 μm.

Figure 4

Spindle disorganization in late-stage oocytes from Klp10A germline mutants. (A) In wild type, a bipolar spindle surrounds the karyosome. Short microtubule fragments are present throughout the ooplasm. (B–G) In oocytes from Klp10A germline mutants, spindles are disorganized. Microtubule fragments in the ooplasm are much longer than wild type and are often arranged in a starburst pattern. (B) Bipolar spindle that is long and frayed. (C) Bipolar spindle in which the two half spindles are not connected by a central spindle. (D) Multipolar spindle. (E and F) Extremely long, disorganized spindles in which the contact between the karyosome and microtubules appears to be lacking. In addition, the spindle in E appears to connect to one of the starburst structures in the ooplasm. (G) Long microtubule fragments in starburst patterns are present throughout the ooplasm of the entire oocyte, not just near the karyosome (arrow). DNA is in blue and microtubules are in green. Bars, 10 μm.

Figure 5

Biorientation of homologous chromosomes is defective in Klp10A germline mutants. (A) In wild type, both the 2nd (red) and 3rd (white) chromosome centromeric FISH probes show biorientation toward opposite spindle poles. All 17 centromere pairs scored were separated (two FISH signals) and oriented correctly. (B) Klp10A germline mutant oocyte prior to or during nuclear envelope breakdown. Homologous centromeres are paired. (C) Klp10A germline mutant oocyte with a long and disorganized spindle, which is nonetheless bipolar and has properly oriented centromeres. (D) Klp10A germline mutant oocyte with a disorganized spindle, loosely “bioriented” chromosomes, but “mono-oriented” 3rd chromosomes. (E and F) Klp10A germline mutant oocytes with disorganized spindles and randomly oriented centromeres. Centromeres in E are not associated with microtubule bundles. DNA is in blue and tubulin is in green. Insets show only the FISH signals. Bars, 10 μm.