Elucidating the mechanism of incomplete cytokinesis and intercellular bridge formation in animal germline cells

提案摘要 本项目的目的是研究不完全胞质分裂导致形成 动物生殖细胞中的细胞间桥。这些桥，在果蝇中被称为环管，是形成 因为这些桥的破坏导致不育。不完全胞质分裂事件不仅广泛见于 生殖细胞，但在一些体细胞组织，是一个典型的霍奇金淋巴瘤的标志，提供了证据， 不完全胞质分裂事件与非种系生物学有关。使用果蝇生殖系作为模型， 不完全的胞质分裂，所提出的实验旨在表征男性环状管的形成， 雌性生殖系，通过生物化学筛选阐明环管膜蛋白质的组成，和 表征从这些筛选中识别出的新型环管组件。该提案的三个综合目标将 提供介导环管形成的细胞骨架和细胞事件的全面理解， 有助于环管形成的蛋白，并评估环管形成所需的新蛋白的功能 在雄性和雌性生殖细胞中。 目标1中提出的实验将定义介导果蝇生殖系中环管形成的细胞事件。 环道形成的机制是未知的，因为没有发表的研究检查环道形成， 在发育中的卵巢或睾丸中的高时间分辨率。此外，环管的组成不同， 男性和女性表明有不同的策略，在性别之间的环形管道的形成。超微结构 环管的研究表明，它们是由膜上的电子密度标记的，然而，这种电子密度的组成部分是由膜上的电子密度标记的。 密度未知。使用活细胞成像和电子断层扫描的组合，环管形成和 将表征膜电子密度的伴随变化。这些实验将是极大的兴趣， 研究生殖细胞生物学的科学家。目标2中提出的实验将鉴定可能促进 环管形成一种邻近依赖的生物素化蛋白质组学方法结合完整环管的分离 复合物将揭示环管复合物的亚结构，并识别蛋白质以进一步验证， 特征化雄性和雌性的蛋白质组将进行比较，从而进一步深入了解它们的共同特征。 生殖系之间的环道形成。在目标3中，将验证在任一筛选中鉴定的候选蛋白， 表征以确定它们是否有助于环管形成的机制。蛋白质定位将首先 使用抗体或荧光标记进行评估，然后进行遗传分析以测试功能。鉴定蛋白质 有助于抑制细胞膜分裂的蛋白质将是研究不完全胞质分裂的研究人员感兴趣的 在生殖系和非生殖系环境中。

Proposal Summary The goal of this project is to investigate the mechanism of incomplete cytokinesis that results in the formation of intercellular bridges in animal germline cells. These bridges, called ring canals in Drosophila, are integral to the formation of gametes as disruption of these bridges results in sterility. Incomplete cytokinesis events are widely observed in not only the germline but in some somatic tissues and are a hallmark of classical Hodgkin lymphoma providing evidence that incomplete cytokinesis events are relevant to non-germline biology. Using the Drosophila germline as a model for incomplete cytokinesis, the experiments proposed are designed to characterize the formation of ring canals in male and female germlines, elucidate the composition of proteins at the ring canal membrane via biochemical screens, and characterize novel ring canal components identified from these screens. The three integrated aims of this proposal will provide a comprehensive understanding of the cytoskeletal and cellular events that mediate ring canal formation, identify proteins that contribute to ring canal formation, and assess the function of novel proteins required for ring canal formation in the male and female germlines. Experiments proposed in Aim 1 will define the cellular events that mediate ring canal formation in the Drosophila germline. The mechanism of ring canal formation is unknown as there are no published studies examining ring canal formation with high-temporal resolution in the developing ovary or testis. Furthermore, the composition of ring canals differs between males and females suggesting there are varying strategies in the formation of ring canals between the sexes. Ultrastructural studies of ring canals reveal they are marked by an electron density at the membrane, however the components of this density are unknown. Using a combination of live cell imaging and electron tomography, ring canal formation and the accompanying changes in membrane electron density will be characterized. These experiments will be of great interest to researchers studying germline biology. Experiments proposed in Aim 2 will identify ring canal proteins that may facilitate ring canal formation. A proximity-dependent biotinylation proteomic approach coupled with isolation of intact ring canal complexes will reveal the substructure of ring canal complexes and identify proteins for further validation and characterization. The proteomes of males and females will be compared, allowing further insight into the shared features of ring canal formation between the germlines. In Aim 3, candidate proteins identified in either screen will be validated and characterized to determine whether they contribute to the mechanism of ring canal formation. Protein localization will first be assessed using antibodies or fluorescent tags, followed by genetic analyses to test for function. Identification of proteins that contribute to the inhibition of membrane abscission will be of interest to researchers studying incomplete cytokinesis in both germline and non-germline contexts.