Control of epithelial morphology and bioenergetics by Toll receptors during dynamic tissue remodeling

项目摘要 在发育过程中，上皮细胞在形态和位置上经历程序性变化，以创造 复杂的组织。在模式生物中的研究已经确定了一组保守的效应蛋白，直接 改变细胞形状，尽管在大细胞群中协调这些过程的上游路径 人们对此仍然知之甚少。研究果蝇上皮细胞重塑的一种范式是细胞嵌入 Neurectoderm，研究表明，高度保守的Toll受体家族的三个成员都有表达 在重叠的条纹图案中组织该组织中的快速细胞重排。通行费受体广泛存在于 在整个人类上皮细胞中表达，并在先天免疫的背景下进行了广泛的研究 发信号。然而，Toll受体对细胞形态的调控却鲜有人关注。焦点 这项建议的目的是了解非均匀Toll受体的表达如何影响皮层张力、细胞-细胞 黏附和线粒体动力学在上皮重塑过程中控制细胞的形状和行为。我们会 使用新开发的CRISPR/Cas9衍生的遗传背景和抗体来表征Toll如何 受体控制细胞极性以触发嵌入；我们将应用非破坏性技术来表征 完整活体胚胎上皮重组的生物能量学；我们将调查未寻址的链接 Toll受体、Rho和G蛋白偶联受体之间的信号转导。我们的第一个假设是邻近的 细胞感觉不同的Toll受体类型表达的差异，以增加皮质张力和 减少细胞与细胞间的黏附。我们已经开发出一种基因系统，可以在单个细胞中表达单个受体 我们将用来系统地表征和比较每种Toll受体类型对细胞的影响 并确定调节细胞形状所需的蛋白质结构域。我们的第二个假设是 在中性胚层伸长过程中细胞的快速重排需要线粒体信号的改变 推动细胞骨架和连接重组。为了测试这一点，我们将使用多光子显微镜来可视化 代谢辅助因子的内源性自发荧光定量检测活体胚胎的细胞氧化还原状态 上皮重塑，然后使用功能获得和丧失技术来确定线粒体在其中的作用 融合和分裂在上皮细胞重组中的作用。我们的第三个假设是Toll受体和GPCR信号 汇聚以激活Rho Kinase，并触发神经外胚层中的细胞嵌入。我们将用得和失- 功能分析以确定这两条信号通路如何相交来控制皮质张力，细胞- 上皮重塑过程中的细胞黏附和线粒体动力学。成功完成这些任务 实验将使我们更全面地了解Toll受体如何在分子水平上发挥作用 在动态组织重塑过程中控制细胞生物力学和生物能量学。

Project Summary During development, epithelial cells undergo programmed changes in morphology and position to create complex tissues. Studies in model organisms have identified a conserved set of effector proteins that directly alter cell shape, although the upstream pathways that coordinate these processes across large groups of cells remain poorly understood. A paradigm for studying epithelial remodeling is cell intercalation in the Drosophila neurectoderm, and it was shown that three members of the highly conserved Toll receptor family are expressed in overlapping striped patterns to organize rapid cell rearrangements in this tissue. Toll receptors are widely expressed throughout human epithelia, and they have been extensively studied in the context of innate immune signaling. However, the control of cell morphology by Toll receptors has received very little attention. The focus of this proposal is to understand how non-uniform Toll receptor expression affects cortical tension, cell-cell adhesion, and mitochondrial dynamics to control cell shape and behavior during epithelial remodeling. We will use newly developed CRISPR/Cas9-derived genetic backgrounds and antibodies to characterize how Toll receptors control cell polarity to trigger intercalation; we will apply non-destructive techniques to characterize the bioenergetics of epithelial reorganization in intact living embryos; and we will investigate unaddressed links between Toll receptor, Rho, and G protein-coupled receptor signaling. Our first hypothesis is that neighboring cells sense differences in the expression of individual Toll receptor types to increase cortical tension and decrease cell-cell adhesion. We have developed a genetic system for expressing individual receptors in a single stripe that we will use to systematically characterize and compare the effects of each Toll receptor type on cell morphology and to identify the protein domains necessary for modulating cell shape. Our second hypothesis is that rapid cellular rearrangements during neurectoderm elongation require changes in mitochondrial signaling to drive cytoskeletal and junctional reorganization. To test this, we will use multiphoton microscopy to visualize the endogenous autofluorescence of metabolic cofactors to quantify cellular redox state in live embryos during epithelial remodeling, and then use gain- and loss-of-function techniques to determine what role mitochondrial fusion and fission play in epithelial reorganization. Our third hypothesis is that Toll receptor and GPCR signaling converge to activate Rho Kinase and trigger cell intercalation in the neurectoderm. We will use gain- and loss- of-functional analyses to determine how these two signaling pathways intersect to control cortical tension, cell- cell adhesion, and mitochondrial dynamics during epithelial remodeling. Successful completion of these experiments will give us a more comprehensive understanding of how Toll receptors function at a molecular level to control cellular biomechanics and bioenergetics during dynamic tissue remodeling.