Investigating novel functions of septate junction proteins during morphogenesis in Drosophila

动物的最终形状是由依赖于形态发生(形态变化)过程的发育事件决定的，包括细胞形状变化和细胞重排。许多对形态发生提供空间和时间控制的机制在所有动物中都是保守的，但对于这些过程是如何工作的仍然缺乏了解。例如，目前对允许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(果蝇)等简单的模式生物中研究这些控制机制有助于理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一起发挥作用。这项研究的重点是这些蛋白质在胚胎发育过程中通过组织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外，该项目还将增加第一代和未被充分代表的少数族裔高中和大学生参与真正的科学研究。具体地说，PI将开发一个新的项目，让来自堪萨斯城的高中生参加为期一个夏天的研究体验，重点是使用模型系统遗传学来研究发展。PI也是堪萨斯大学几个旨在增加未被充分代表的群体参与科学的项目的导师，包括教授一门关于发展应用于STEM学科研究生院的技能的课程。形态发生事件由发育调节的信号通路触发和控制，这些信号通路导致单个细胞中的肌动球蛋白动态。然后，这些力量必须通过上皮传播并保持，以便引起适当的组织水平的形态变化。虽然人们对肌动球蛋白的动力学了解很多，但对组织中的力传播所涉及的细胞机制却知之甚少。在初步研究中，PI确定苍蝇的胚胎形态发生事件需要大量编码核心间隔连接(SJ)成分的基因集合。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，这表明这些蛋白在形态发生中具有新的功能。这个项目的总体目标是从机制上理解SJ蛋白如何促进背部闭合(DC)的形态发生过程，DC是一种广泛研究的发育事件，发生在果蝇胚胎发生的中期。我们将致力于以下目标：(1)确定SJ蛋白是否调节DC过程中肌动球蛋白复合体的组装和动态；(2)确定DC过程中SJ蛋白是否是生物力学或黏附功能所必需的；(3)确定SJ蛋白是否需要维持顶端/基底端的极性。肌球蛋白动力学将通过实时成像和带有抗体的固定组织来检测，这些抗体揭示了激活的肌球蛋白复合体，而生物力学特性将通过激光显微外科实验来探索。

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.