Endow S A, Komma D J
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
J Cell Sci. 1996 Oct;109 ( Pt 10):2429-42. doi: 10.1242/jcs.109.10.2429.
The Ncd microtubule motor protein is required for meiotic and early mitotic chromosome distribution in Drosophila. Null mutant females expressing the Ncd motor fused to the Aequorea victoria green fluorescent protein (GFP), regulated by the wild-type ncd promoter, are rescued for chromosome segregation and embryo viability. Analysis of mitosis in live embryos shows cell cycle-dependent localization of Ncd-GFP to centrosomes and spindles. The distribution of Ncd-GFP in spindles during metaphase differs strikingly from that of tubulin: the tubulin staining is excluded by the chromosomes at the metaphase plate; in contrast, Ncd-GFP forms filaments along the spindle microtubules that extend across the chromosomes. The existence of Ncd-GFP fibers that cross the metaphase plate suggests that Ncd interacts functionally with chromosomes in metaphase. Differences are no longer observed in anaphase when the chromosomes have moved off the metaphase plate. A mutant form of Ncd fused to GFP also localizes to spindles in live embryos. Mutant embryos show frequent centrosome and spindle abnormalities, including free centrosomes that dissociate from interphase nuclei, precociously split centrosomes, and spindles with microtubule spurs or bridges to nearby spindles. The precociously split and free centrosomes indicate that the Ncd motor acts in cleavage stage embryos to maintain centrosome integrity and attachment to nuclei. The frequent spindle spurs of mutant embryos are associated with mis-segregating chromosomes that partially detach from the spindle in metaphase, but can be recaptured in early anaphase. This implies that the Ncd motor functions to prevent chromosome loss by maintaining chromosome attachment to the spindle in metaphase, consistent with the Ncd-GFP fibers that across the metaphase plate.
Ncd微管运动蛋白对于果蝇减数分裂和早期有丝分裂染色体分布是必需的。由野生型ncd启动子调控、表达与维多利亚多管水母绿色荧光蛋白（GFP）融合的Ncd运动蛋白的无效突变雌性果蝇，在染色体分离和胚胎活力方面得到了挽救。对活胚胎有丝分裂的分析表明，Ncd-GFP在细胞周期中定位于中心体和纺锤体。中期时，Ncd-GFP在纺锤体中的分布与微管蛋白的分布显著不同：中期板处的染色体排除了微管蛋白染色；相反，Ncd-GFP沿着纺锤体微管形成细丝，这些细丝延伸穿过染色体。穿过中期板的Ncd-GFP纤维的存在表明，Ncd在中期与染色体在功能上相互作用。当染色体离开中期板时，后期不再观察到差异。与GFP融合的Ncd突变形式也定位于活胚胎的纺锤体。突变胚胎显示出频繁的中心体和纺锤体异常，包括从间期核解离的游离中心体、过早分裂的中心体以及带有微管刺或与附近纺锤体相连的桥的纺锤体。过早分裂和游离的中心体表明，Ncd运动蛋白在卵裂期胚胎中起作用，以维持中心体的完整性以及与细胞核的附着。突变胚胎中频繁出现的纺锤体刺与中期时部分从纺锤体脱离但在早后期可重新捕获的染色体错分有关。这意味着Ncd运动蛋白通过在中期维持染色体与纺锤体的附着来防止染色体丢失，这与穿过中期板的Ncd-GFP纤维一致。
