STUDIES ON OVARIAN RING CANALS IN DROSOPHILA

这项工作的长期目标是了解称为环管的细胞间连接如何促进合胞发育的实例。环管起源于被细胞骨架蛋白边缘稳定的有丝分裂分裂沟。它们普遍存在于整个动物王国的生殖细胞中，也存在于许多体细胞中。然而，在大多数情况下，环管的功能意义尚不清楚。由于环管发育缺陷导致细胞事件协调的中断可能导致细胞行为的严重畸变，如失去生长控制。实验系统是果蝇的卵发生，在此过程中，护理细胞通过环管将细胞质转移到卵母细胞，为正在生长的卵母细胞提供母体成分。通过研究细胞质运输缺陷导致雌性不育的突变蝇株，已经确定了几个环管成分。在这项拨款申请中提出的工作重点是这些蛋白质的功能分析。该提案的具体目的是：(1)鉴定在环管形态发生过程中与丝蛋白（由cheerio编码）相互作用的蛋白质；(2) HTS基因座编码蛋白的功能分析及HTS蛋白加工研究；(3) kelch蛋白功能分析及kelch相互作用蛋白鉴定。果蝇卵室的环管有一个基于肌动蛋白的细胞骨架，它保持环管的完整性，并允许环管在癌基因期间直径增长超过10倍。细胞骨架与环管质膜连接的机制尚不清楚。丝蛋白是提供这种连接的一个很好的候选物，并且提出了在卵腔中鉴定与丝蛋白相互作用的蛋白质的研究。这将提供从解理沟到环形管过渡的信息。hts位点在卵泡细胞和生殖细胞中都是必需的。在生殖系中，其产物存在于环管和丝体中。aim 2的研究主要集中在描述融合体行为和环管组装之间的联系。最后，环管的肌动蛋白细胞骨架是不寻常的，因为细丝排列成一个圆圈。kelch蛋白在环管生长过程中对维持肌动蛋白的包装组织起着重要作用。在目标3中，提出了定义kelch的生化作用的工作。这项工作将包括使用冷冻电子显微镜对野生型和kelch突变型卵室中的肌动蛋白组织进行高分辨率研究。

The long-term goal of this work is to understand how intercellular junctions called ring canals contribute to instances of syncytial development. Ring canals originate as arrested mitotic cleavage furrows that become stabilized by a rim of cytoskeletal protein. They are prevalent in developing germ cells throughout the animal kingdom and have also been found in a number of somatic cells. However, the functional significance of ring canals is poorly understood in most of these cases. Disruptions in the coordination of cellular events as a result of faulty ring canal development are likely to cause severe aberrations in cell behavior such as loss of growth control. The experimental system is Drosophila oogenesis, during which nurse cells feed growing oocytes maternal components by transferring cytoplasm to the oocyte through ring canals. By studying mutant fly strains in which defective transport of cytoplasm leads to female sterility, several ring canal components have been identified. The work proposed in this grant application is focused on functional analysis of these proteins. The specific aims of the proposal are: (1) identification of proteins that interact with filamin (encoded by cheerio) during ring canal morphogenesis; (2) functional analysis of proteins encoded by the hts locus and studies on hts protein processing; (3) analysis of kelch protein function and identification of kelch interacting proteins. The ring canals of Drosophila egg chambers have an actin-based cytoskeleton that maintains ring canal integrity and also allows ring canals to grow over 10-fold in diameter during oncogenes. The mechanism for linking the cytoskeleton to the plasma membrane of ring canals is unknown. Filamin is a good candidate for providing such a linkage, and studies are proposed to identify proteins that interact with filamin in egg chambers. This should provide information on the transition from cleavage furrow to ring canal. The hts locus is required in both follicle cells and germline cells. In the germline, hts products are found in both ring canals and the fusome. Studies in aim 2 are focused on characterizing the link between fusome behavior and ring canal assembly. Finally, the actin cytoskeleton of ring canals is unusual because the filaments are arrange in a circle. The kelch protein is important for maintaining the packing organization of actin during the growth of ring canals. In aim 3, work is proposed to define the biochemical role of kelch. The work will include high resolution studies of actin organization in wild type and kelch mutant egg chambers using cryoelectron microscopy.