The growth of the germline ring canals during Drosophila melanogaster oogenesis

项目摘要 不孕不育和生育能力受损每年影响数百万人；因此，了解这些途径和 调节正常配子形成的机制是必要的。细胞间桥是必不可少的结构 发现于整个动物界发育中的精子和卵子；它们连接邻近的细胞，使 材料共享和行为协调。该项目的长期目标是了解如何 细胞间桥形成、稳定和扩张。生殖系细胞间桥，或环 在发育中的果蝇卵室中发现的沟渠已经成为首要的模型系统。在之后形成 不完全胞质分裂，生殖系环管经历20倍的扩张以促进材料的转移 从支持哺育细胞到发育中的卵母细胞。影响环管形成、稳定性或 肥大会导致不孕。许多结构和调节蛋白定位于环管和/或调节 它们的结构方面；然而，缺乏连接蛋白质和通路的完整模型。这个 Pi的实验室表征了Ste20激酶、畸形蛋白(MSN)、SH2/SH3接头蛋白、DREADLOCK的作用 (DOCK)，以及两个肌动蛋白核仁的协同活性，Arp2/3复合体和福尔明，透明的 (Dia)，在环管大小和稳定性的调节中；然而，其背后的确切机制 这些贡献以及这些监管机构之间的联系尚不清楚。这项建议的目的是 确定这些蛋白质如何促进环管的形成、稳定和扩张，以及它们的活性 是相互整合的，并与其他已知的环管蛋白整合在一起。中心假设是MSN，Dock， Dia和Arp2/3复合体在空间和时间上协调粘着连接蛋白的内吞作用， 肌球蛋白的活性，并改变肌动蛋白的细胞骨架，以促进环管扩张和维持稳定。 目的1将验证一种假说，即黏附连接蛋白E-钙粘附素调节内吞作用 环形渠道的大小和稳定性。目标2将确定Arp2/3复合体和/或Dia是否间接调节环 通过影响粘连连接或肌球蛋白活性来扩张根管。AIM 3将使用基于候选人的 鉴定协调肌动蛋白细胞骨架变化和细胞黏附的Dock相互作用蛋白的方法 在生殖系中。课程将结合使用适合本科生的技术，包括荧光技术。 活体和固定样品的成像、定量图像分析、上位性实验和基础生物化学。 PI在指导25名本科生研究人员方面的出色记录，她在细胞和 发育生物学，以及果蝇模型系统的强大和可获得性，使她处于有利地位 完成提议的目标。通过参与这些目标，学生将获得宝贵的培训和 这些经验将增加他们对STEM领域的兴趣。正在研究的蛋白质、结构和过程 在正常的形态发生过程中也会被利用，在疾病中可能会被错误地调节；因此，从这一点上的见解 这项工作将提高我们对人类正常发育和疾病的理解。

Project Summary Infertility and impaired fecundity affect millions of people each year; therefore, understanding the pathways and mechanisms that regulate normal gamete formation are necessary. Intercellular bridges are essential structures found in developing sperm and eggs across the animal kingdom; they connect neighboring cells and allow the sharing of materials and coordination of behaviors. The long-term goal of this project is to understand how intercellular bridges are formed, stabilized, and undergo expansion. The germline intercellular bridges, or ring canals, found in the developing fruit fly egg chamber have emerged as the premier model system. Formed after incomplete cytokinesis, the germline ring canals undergo a 20-fold expansion to facilitate the transfer of materials from the supporting nurse cells to the developing oocyte. Mutations that affect ring canal formation, stability, or expansion lead to infertility. Many structural and regulatory proteins localize to the ring canals and/or regulate aspects of their structure; however, an integrated model connecting the proteins and pathways is lacking. The PI’s lab characterized a role for the Ste20 kinase, Misshapen (Msn), the SH2/SH3 adaptor protein, Dreadlocks (Dock), and the coordinated activity of two actin nucleators, the Arp2/3 complex and the formin, Diaphanous (Dia), in the regulation of ring canal size and stability; however the precise mechanisms underlying their contribution and the connections between these regulators is not known. The objective of this proposal is to determine how these proteins contribute to ring canal formation, stability, and expansion, and how their activity is integrated with each other and with other known ring canal proteins. The central hypothesis is that Msn, Dock, Dia, and the Arp2/3 complex spatially and temporally coordinate endocytosis of adherens junction proteins, myosin activity, and changes to the actin cytoskeleton to promote ring canal expansion and maintain stability. Aim 1 will test the hypothesis that regulated endocytosis of the adherens junction protein, E-Cadherin, regulates ring canals size and stability. Aim 2 will determine whether the Arp2/3 complex and/or Dia indirectly regulate ring canal expansion through effects on adherens junctions or myosin activity. Aim 3 will use a candidate-based approach to identify Dock-interacting proteins that coordinate changes in the actin cytoskeleton and cell adhesion in the germline. A combination of undergraduate-appropriate techniques will be used, including fluorescence imaging of live and fixed samples, quantitative image analysis, epistasis experiments, and basic biochemistry. The PI’s strong track record of mentoring 25 undergraduate student researchers, her expertise in cell and developmental biology, and the power and accessibility of the fruit fly model system make her well-positioned to complete the proposed aims. Through their involvement in these aims, students will gain valuable training and experience that will increase their interest in STEM fields. The proteins, structures, and processes being studied are also utilized during normal morphogenesis and can be mis-regulated in disease; therefore, insight from this work will improve our understanding of both normal development and disease in humans.