Investigating novel functions of septate junction proteins during morphogenesis in Drosophila

研究果蝇形态发生过程中隔膜连接蛋白的新功能

• 批准号: 1656835
• 负责人: Robert Ward
• 金额: $ 70万
• 依托单位: University of Kansas Center for Research Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1656835&HistoricalAwards=false
• 关键词: Investigating; novel; functions; septate; junction

The final shape of an animal is determined by developmental events that rely on morphogenetic (change in form) processes including cell shape changes and cell rearrangements. Many of the mechanisms that provide spatial and temporal control over morphogenesis are conserved in all animals, yet knowledge is still lacking on how these processes work. For example, the current understanding of mechanisms that allow mechanical forces to propagate through a tissue is inadequate to fully explain morphogenesis in many developmental events. Thus investigating these control mechanisms in a simple model organism such as the fruit fly (Drosophila melanogaster) provide understanding of basic developmental processes that are shared by all animals. The Ward lab has identified a number of proteins that appear to function together in several morphogenetic events during embryogenesis in the fly. This research focuses on the molecular functions of these proteins in generating and propagating mechanical forces through a tissue during embryogenesis. In addition to the scientific advancements that will result from this study, this project will increase the participation of first generation and underrepresented minority high school and college students in authentic scientific research. Specifically, the PI will develop a new program to engage high school students from Kansas City, KS in a summer-long research experience centered on using model system genetics to study development. The PI is also a mentor for several programs aimed at increasing the participation of underrepresented groups in the sciences at the University of Kansas, including teaching a course on developing skills to apply to graduate school in STEM disciplines.Morphogenetic events are triggered and controlled by developmentally-regulated signaling pathways that lead to actomyosin dynamics in individual cells. These forces must then be propagated through the epithelium and maintained in order to elicit appropriate tissue-level morphological changes. Although much is known about actomyosin dynamics, less is known about the cellular mechanisms involved in force propagation through a tissue. In preliminary studies, the PI determined that a large collection of genes that encode core septate junction (SJ) components are required for embryonic morphogenetic events in the fly. The SJ is a large protein complex that functions as an occluding junction. Importantly, the requirement for these genes occurs prior to the formation of the occluding junction, indicating a novel function for these proteins in morphogenesis. The overall goal of this project is to provide mechanistic understanding of how SJ proteins facilitate morphogenetic processes during dorsal closure (DC), a well-studied developmental event that occurs midway through Drosophila embryogenesis. The following aims will be addressed: (1) to determine if SJ proteins regulate the assembly and dynamics of actomyosin complexes during DC, (2) to determine if SJ proteins are required for biomechanical or adhesive functions during DC, and (3) to determine if SJ proteins are required to maintain apical/basal polarity. Actomyosin dynamics will be examined by live imaging and on fixed tissues with antibodies that reveal activated myosin complexes, while biomechanical properties will be probed by laser microsurgery experiments.

动物的最终形状是由依赖于形态发生（形式变化）过程的发育事件决定的，这些过程包括细胞形状变化和细胞重排。许多提供形态发生的空间和时间控制的机制在所有动物中都是保守的，但关于这些过程如何工作的知识仍然缺乏。例如，目前对机械力在组织中传播的机制的理解不足以完全解释许多发育事件中的形态发生。因此，在果蝇（Drosophila melanogaster）等简单模式生物中研究这些控制机制，有助于了解所有动物共有的基本发育过程。沃德实验室已经确定了一些蛋白质，这些蛋白质似乎在果蝇胚胎发生过程中的几个形态发生事件中共同起作用。本研究的重点是这些蛋白质在胚胎发生过程中产生和传播机械力的分子功能。除了这项研究将带来的科学进步之外，该项目还将增加第一代和未被充分代表的少数民族高中生和大学生对真正科学研究的参与。具体来说，PI将开发一个新项目，让堪萨斯城的高中生参与一个为期一个夏天的研究项目，重点是使用模型系统遗传学来研究发育。PI还是几个项目的导师，这些项目旨在提高堪萨斯大学（University of Kansas）未被充分代表的群体在科学领域的参与度，包括教授一门关于培养技能以应用于STEM学科研究生院的课程。形态发生事件是由发育调节的信号通路触发和控制的，这些信号通路导致单个细胞中的肌动球蛋白动力学。然后这些力必须通过上皮传播并维持，以引起适当的组织水平形态变化。虽然对肌动球蛋白动力学了解很多，但对组织中力传播的细胞机制知之甚少。在初步研究中，PI确定了大量编码核心分隔结（SJ）成分的基因是果蝇胚胎形态发生事件所必需的。SJ是一个大的蛋白质复合体，起着闭塞连接的作用。重要的是，对这些基因的需求发生在闭塞连接形成之前，表明这些蛋白质在形态发生中的新功能。该项目的总体目标是提供SJ蛋白如何促进背侧闭合（DC）过程的机制理解，DC是发生在果蝇胚胎发生中途的一个充分研究的发育事件。以下目标将被解决：(1)确定SJ蛋白是否在DC期间调节肌动球蛋白复合物的组装和动力学，(2)确定SJ蛋白是否在DC期间需要生物力学或粘附功能，以及(3)确定SJ蛋白是否需要维持尖/基极性。肌动球蛋白动力学将通过实时成像和固定组织上的抗体来检测，抗体显示激活的肌球蛋白复合物，而生物力学特性将通过激光显微手术实验来探测。