STUDIES ON OVARIAN RING CANALS IN DROSOPHILA

这项工作的长期目标是了解被称为环管的细胞间连接如何有助于合体发育。环管起源于受阻的有丝分裂沟，分裂沟被细胞骨架蛋白的边缘稳定。它们普遍存在于整个动物界发育中的生殖细胞中，也在许多体细胞中发现。然而，在大多数情况下，对环状管道的功能意义知之甚少。由于环管发育不良导致细胞活动协调中断，可能会导致细胞行为严重异常，如失去生长控制。实验系统是果蝇卵子发生，在此过程中，哺育细胞通过环形通道将细胞质转移到卵母细胞，从而喂养正在生长的卵母细胞。通过研究细胞质运输缺陷导致雌性不育的突变苍蝇品系，已经鉴定出几种环管成分。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细丝蛋白(由cheiio编码)相互作用的蛋白质；(2)HTS基因座编码的蛋白质的功能分析和HTS蛋白加工研究；(3)海带蛋白功能的分析和海带相互作用蛋白的鉴定。果蝇卵室的环管有一个以肌动蛋白为基础的细胞骨架，它保持环管的完整性，并允许环管在致癌过程中直径扩大10倍以上。将细胞骨架连接到环管的质膜的机制尚不清楚。细丝是提供这种联系的一个很好的候选者，有人建议进行研究，以确定在卵室中与细丝相互作用的蛋白质。这应该提供从卵裂沟向环沟过渡的信息。HTS基因在卵泡细胞和生殖细胞中都是必需的。在胚系中，HTS产物存在于环管和梭形体中。在目标2中的研究集中于描述梭形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，因为这些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堆积组织起重要作用。在目标3中，提出了定义海带的生化作用的工作。这项工作将包括使用冷冻电子显微镜对野生型和海带突变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.