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，加上果蝇卓越的遗传学和细胞学，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相协调来产生有功能的精子。通过突变分析，我们已经确定ASSON(ASUN)是果蝇精子发生的重要调节因子，它编码一种进化上保守的蛋白质。ASun精母细胞在减数分裂前期停滞。引人注目的是，停滞的精母细胞含有不能稳定地与细胞核结合的游离型中心体。克服停滞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ASun减数分裂后的精子细胞中，中心粒衍生的基体从细胞核中分离出来，导致后来的精子发生异常。我们发现ASun精母细胞和精子细胞表现出核周动力蛋白的急剧减少。动力蛋白是一种负端导向的微管运动复合体，是从细胞内货物运输到细胞迁移等多种生物学过程所必需的。动力蛋白在多个水平上受到控制，包括对其亚细胞定位的调节；然而，动力蛋白靶向于细胞内不同部位的机制尚不清楚。我们目前的模型是，ASun通过促进动力蛋白募集到核表面来协调精子发生，这是M期进入核-中心体耦合和减数分裂保真所必需的关键步骤。在果蝇雄性减数分裂过程中，ASUN呈现动态定位模式，其从细胞核释放到细胞质的时间与G2精母细胞中细胞核表面动力蛋白的出现有关。我们将评估精母细胞内这种受调控的ASun运动是否对控制动力蛋白的活性至关重要。我们提出的实验将使我们能够更详细地了解动力蛋白被招募到核表面的机制，重点是阐明ASun在这一过程中的作用。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优先结合到翻译后通过乙酰化修饰的微管上。我们将验证我们的假设，即这一乙酰化微管池介导了果蝇雄性减数分裂的关键事件，包括核-中心体耦合。拟议的实验有可能阐明ASun的作用机制，确定动力蛋白马达复合体募集到核表面所需的额外因素，并确定微管乙酰化在精子发生中的作用。

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.