Developmental Regulation of the Cell Cycle in Drosophila

描述（申请人提供）：参与细胞周期的基因的高度功能保守性与黑腹果蝇的极好遗传学和细胞学相结合，使其成为研究发育背景下细胞周期调控的理想模式生物。精子发生利用有丝分裂和减数分裂细胞周期与生长和分化程序协调来产生功能性精子。通过突变分析，我们已经确定asunder（asun），它编码一个进化上保守的蛋白质，作为果蝇精子发生的重要调控因子。在减数分裂Ⅰ前期，精子发生阻滞。引人注目的是，被捕的精母细胞含有游离的中心体，不能稳定地与细胞核结合。克服停滞期的精母细胞在减数分裂纺锤体组装、染色体分离和胞质分裂方面表现出严重的缺陷。此外，在未成熟的减数分裂后精子细胞中，中心粒衍生的基体从细胞核中分离，导致精子发生的后期异常。我们发现asun精母细胞和精子细胞的核周动力蛋白明显减少。动力蛋白是一种负末端导向的微管运动复合物，它是从细胞内货物运输到细胞迁移等多种生物过程所必需的。动力蛋白在多个水平上受到控制，包括调节其亚细胞定位;然而，动力蛋白靶向细胞内不同位点的机制还不清楚。我们目前的模型是，asun协调精子发生，促进动力蛋白招聘到核表面，一个关键的步骤，需要在M期进入核-中心体耦合和减数分裂的保真度。ASUN在果蝇雄性减数分裂过程中呈现动态定位模式，其从细胞核释放到细胞质的时间与G2精母细胞核表面动力蛋白的出现相关。我们将评估是否这种调节运动的精母细胞内的ASUN是至关重要的控制动力蛋白的活性。我们提出的实验，将使我们能够获得一个更详细的了解的机制，动力蛋白被招募到核表面的重点是阐明ASUN在这个过程中的作用。我们的初步数据表明，锚定在精母细胞核表面的动力蛋白可能会优先结合到微管后，通过乙酰化修饰。我们将测试我们的假设，这个池的乙酰化微管介导的关键事件的果蝇雄性减数分裂，包括核-中心体耦合。拟议的实验有可能阐明ASUN的作用机制，以确定额外的因素所需的招募的动力蛋白运动复合物的核表面，并定义在精子发生过程中的微管乙酰化的作用。 公共卫生相关性：控制细胞内动力蛋白马达定位的机制尚不清楚。我们已经表明，ASUN是所需的招募动力蛋白的果蝇精母细胞的核表面，减数分裂的保真度的一个关键事件。由于脊椎动物同源的ASUN也控制动力蛋白的本地化在培养的人类细胞有丝分裂过程中，阐明的作用机制的ASUN可能有重要意义的人类疾病，如癌症。

DESCRIPTION (provided by applicant): The high degree of functional conservation of genes involved in the cell cycle combined with the superb genetics and cytology of Drosophila melanogaster make it an ideal model organism for studying cell-cycle regulation in a developmental context. Spermatogenesis utilizes mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. By mutational analysis, we have identified asunder (asun), which encodes an evolutionarily conserved protein, as an essential regulator of Drosophila spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus. Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein. Dynein is a minus end-directed microtubule motor complex that is required for diverse biological processes, from transport of intracellular cargo to cell migration. Dynein is controlled at multiple levels, including regulation of its subcellular localization; the mechanisms underlying the targeting of dynein to various sites within cells, however, are not well understood. Our current model is that asun coordinates spermatogenesis by promoting dynein recruitment to the nuclear surface, a critical step that is required for nucleus-centrosome coupling at M-phase entry and fidelity of meiotic divisions. ASUN exhibits a dynamic localization pattern during Drosophila male meiosis, and the timing of its release from the nucleus to the cytoplasm correlates with the appearance of dynein on the nuclear surface in G2 spermatocytes. We will assess whether this regulated movement of ASUN within spermatocytes is critical for controlling the activity of dynein. We propose experiments that will allow us to gain a more detailed understanding of the mechanism by which dynein is recruited to the nuclear surface with a focus on elucidating the role of ASUN in this process. Our preliminary data suggest that dynein anchored on the nuclear surface of spermatocytes may preferentially bind to microtubules that are post-translationally modified by acetylation. We will test our hypothesis that this pool of acetylated microtubules mediates key events of Drosophila male meiosis, including nucleus-centrosome coupling. The proposed experiments have the potential to illuminate the mechanism of action of ASUN, to identify additional factors required for recruitment of the dynein motor complex to the nuclear surface, and to define the role of microtubule acetylation during spermatogenesis. PUBLIC HEALTH RELEVANCE: The mechanisms controlling localization of dynein motors within cells are not well understood. We have shown that ASUN is required for recruitment of dynein to the nuclear surface of Drosophila spermatocytes, a critical event for fidelity of meiotic divisions. Because the vertebrate homolog of ASUN also controls dynein localization in cultured human cells during mitosis, elucidation of the mechanism of action of ASUN may have important implications for human diseases such as cancer.