Spatial Dynamics of Phosphorylation on the Regulatory Light Chains of Myosin II in Migrating Cells

这个项目的广泛科学目标是了解细胞在组织发育和中生动物体内稳态过程中是如何爬行的。由于内皮细胞的运动在血管的形成和修复中的重要性，因此将对构成血管衬里的内皮细胞进行研究。然而，其他组织细胞执行类似的运动，这些运动对于广泛的生物学过程是必不可少的，包括胚胎发育、器官形成、组织修复、免疫反应和其他过程。该项目将研究肌球蛋白II，这是一种在协调爬行运动中发挥重要作用的运动蛋白。肌球蛋白II可以收缩细胞质，定向细胞延伸，并产生力量将细胞向前拉。这些活动必须在准确的位置和适当的时间发生，才能使细胞发生定向运动，但肌球蛋白II在细胞运动过程中的行为如何受到控制还不是很清楚。特别是，控制活细胞质中肌球蛋白II活性的信号的位置和时间尚不清楚。实验将在爬行细胞中确定肌球蛋白II调节轻链上磷酸化的动力学和空间分布，这种修饰已知会影响分离的肌球蛋白II的活性。显微镜观察和生化分析将结合生化分析来确定在细胞培养模型中，当牛主动脉内皮细胞爬行修复小伤口时，肌球蛋白II何时何地被磷酸化。放射性磷酸盐和识别磷酸化肌球蛋白II的抗体将被用来监测磷酸化的动力学和位置，还将构建一种修饰形式的肌球蛋白II，它被标记有一种染料，这种染料会随着磷酸化而改变。标记的肌球蛋白II将允许在活细胞中直接在显微镜下观察到磷酸化，因此可以根据细胞运动的精确关系来观察生化变化的时间和位置。肌球蛋白II上磷酸化位点的遗传改变，以及影响蛋白质磷酸化的药物，将被用来操纵迁移细胞中肌球蛋白II磷酸化的动力学，以确定磷酸化对肌球蛋白II在体内行为的影响程度，进而确定肌球蛋白II调节如何影响细胞运动。智力优势：通过解剖活细胞胞浆中肌球蛋白II磷酸化的动力学，这项工作将为调节细胞运动机制的分子基础提供新的和基本的信息。将磷酸化作为肌球蛋白II行为的一种潜在调节机制的研究将明确地测试一种被广泛引用但仍在很大程度上不确定的关于细胞质收缩如何被控制的解释。这将大大提高我们对细胞如何爬行的理解，这是一个普遍的、基本的生物过程。广泛的影响：分子生物化学和细胞行为之间的接口是一个日益增长的重要感兴趣领域，在组织工程等领域具有潜在的社会利益。该项目还将有助于对学生进行基础研究的培训。

The broad scientific objective of this project is to understand how cells crawl during tissue development and homeostasis in mesozoans. Endothelial cells, which form the lining of blood vessels, will be studied because of the importance of their movements in the formation and repair of blood vessels. However, other tissue cells perform similar movements that are essential to a vast range of biological processes, including embryonic development, organ formation, tissue repair, the immune response, and other processes. The project will examine myosin II, a motor protein that plays an important role in coordinating crawling movements. Myosin II can contract cytoplasm, orient cell extensions, and generate forces to pull a cell forward. These activities must occur in precise locations and at appropriate times in order for directed cell movement to occur, but how myosin II behavior is controlled during cell movement is not well understood. In particular, the location and timing of the signals that control myosin II activity in living cytoplasm are unclear. Experiments will be performed to determine, in crawling cells, the kinetics and spatial distribution of phosphorylation on the regulatory light chains of myosin II, a modification known to affect the activity of isolated myosin II. Microscopic observations will be coupled with biochemical analysis to determine when and where myosin II is phosphorylated in bovine aortic endothelial cells as they crawl to heal small wounds in a cell culture model. Radioactive phosphate and antibodies that recognize phosphorylated myosin II will be used to monitor the kinetics and location of phosphorylation, and a modified form of myosin II that is labeled with a dye that changes in response to phosphorylation will also be constructed. The labeled myosin II will allow phosphorylation to be observed directly under a microscope in living cells, so that the timing and location of biochemical changes can be viewed in precise relationship to cell movements. Genetic alteration of the phosphorylation sites on myosin II, along with pharmacological agents that affect protein phosphorylation, will then be used to manipulate the kinetics of myosin II phosphorylation in migrating cells, in order to determine the extent to which phosphorylation affects myosin II behavior in vivo and, in turn, how myosin II regulation influences cell movement.Intellectual merit: By dissecting the dynamics of myosin II phosphorylation in the cytoplasm of living cells, this work will provide new and essential information about the molecular basis for regulation of the cellular locomotive machinery. Examination of phosphorylation as a potential regulatory mechanism of myosin II behavior will definitively test a widely invoked, but still largely uncertain, explanation for how cytoplasmic contractility is controlled. This will significantly advance our understanding of how cells crawl, which is a universal, fundamental biological process.Broader impacts: The interface between molecular biochemistry and cellular behavior is a growing and vital area of interest that has potential societal benefits in areas such as tissue engineering. This project will also contribute to the training of students in basic research.