Control of cell ratcheting engagement during epithelial morphogenesis

上皮形态发生过程中细胞棘轮啮合的控制

• 批准号: 10366809
• 负责人: James Todd Blankenship
• 金额: $ 29.49万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366809
• 关键词: 1-Phosphatidylinositol 3-Kinase; Actomyosin; Adopted; Adult; Apical; Area; Behavior; Binding; Biochemical; Biological; Biological Assay; Cell Aging; Cell Shape; Cell membrane; Cell physiology; Cell surface; Cells; Contracts; Coupled; Cues; Data; Data Analyses; Development; Dimensions; Drosophila genome; Drosophila genus; Embryo; Epithelial; Epithelial Cells; FRAP1 gene; Frequencies; Generations; Goals; Image; Individual; Intention; Intercalated Cell; Lead; Lipids; Mass Spectrum Analysis; Measurement; Measures; Membrane; Mesoderm; Mitochondria; Morphogenesis; Morphology; Nature; PH Domain; PIK3CG gene; Pathway interactions; Pattern; Periodicity; Phase; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphotransferases; Physiologic pulse; Population; Process; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Reporting; Signal Pathway; Signal Transduction; Surface; System; Testing; Tissues; Tubular formation; Work; base; cell cortex; cell motility; constriction; course development; gastrulation; in vivo; insight; intercalation; interfacial; mTOR protein; novel; phosphatidylinositol 3,4,5-triphosphate; protein function; recruit; wound healing

Project Summary Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating high-tension contractile forces at the cell cortex before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be cellular processes to extract unidirectional changes that drive processive transformations in cell shape. In previous work (Jewett et al., 2017; Miao et al., 2019), we found that cytoskeletal force generation is coordinated with endocytic remodeling of the plasma membrane through Sbf- Rab35 tubular compartmental function to stabilize contracted cell surfaces and permit the shrinking of cell apices (apical constriction) or cell interfaces (cell intercalation). However, how this membranous cellular ratchet becomes engaged at particular cell surfaces remains unclear. In the proposed studies, we will examine the informational signals that engage ratcheting and direct Sbf/Rab35 compartmental behaviors to contracting interfaces or cell apices, and identify the fundamental changes in oscillatory durations, amplitudes, frequencies, and/or directionality that lead to contractile processivity. Our preliminary data indicates the PIP3 is a critical determinant for ratcheting engagement – through our proposed work we will perform the first characterization of phosphatidylinositol phosphates (PIPs) in providing lipid-based membrane cues for morphogenesis and gastrulation/ratcheting dynamics in the early Drosophila embryo. In the first aim, we will also analyze how the plasma membrane ultrastructure is remodeled by ratcheting processes and determine if PIP levels are developmentally patterned to drive apical constriction during mesoderm ingression. Our project then moves to a systematic identification of Sbf and Rab35 protein partners in directing ratcheting engagement, and examines the cell signaling pathways that direct a “switching” behavior of contractile force generation from the apical surface to cell interfaces. Our data indicates that, in the absence of JAK/STAT signaling, the Sbf-Rab35 ratchet becomes engaged on all apical surfaces in the embryo, resulting in global apical flattening and constriction. Further, our studies will define if a larger Upd-JAK-STAT-Pi3K-PIP3-Sbf- Rab35 pathway or if two independent pathways (PIP3 and JAK/STAT) have been coordinated to regulate ratcheting engagement. We also apply a new computational phase-based osculating circle approach to detect active periods of contraction and expansion displacements. Finally, we are developing a new mito-tag ectopic relocalization assay as a measure of “sufficiency” of recruiting factors in vivo, and examine if the Akt/mTOR pathway regulates cell ratcheting, potentially demonstrating a new, highly novel function of Akt/mTOR in controlling epithelial cell topologies. Thus, the planned project has the potential to elucidate a large, regulatory hierarchy of the mechanisms that guide engagement of cell ratcheting in epithelial tissues.

项目概要 上皮组织中产生的力通常是脉动的，肌动球蛋白网络产生高压 循环分解之前细胞皮层的收缩力。细胞骨架力的脉冲性质 意味着必须存在细胞过程来提取驱动进行性的单向变化 细胞形状的转变。在之前的工作中（Jewett et al., 2017; Miao et al., 2019），我们发现 细胞骨架力的产生通过 Sbf- 与质膜的内吞重塑相协调 Rab35 管状区室功能可稳定收缩的细胞表面并允许细胞收缩 顶端（顶端收缩）或细胞界面（细胞嵌入）。然而，这种膜细胞如何 棘轮与特定细胞表面的啮合仍不清楚。在拟议的研究中，我们将 检查参与棘轮效应并指导 Sbf/Rab35 区室行为的信息信号 收缩界面或细胞顶端，并识别振荡持续时间、振幅、 导致收缩持续性的频率和/或方向性。我们的初步数据表明 PIP3 是 棘轮参与的关键决定因素——通过我们提议的工作，我们将执行第一个 磷脂酰肌醇磷酸盐 (PIP) 的表征，为细胞提供基于脂质的膜线索 早期果蝇胚胎的形态发生和原肠胚形成/棘轮动力学。在第一个目标中，我们将 还分析质膜超微结构如何通过棘轮过程重塑，并确定是否 PIP 水平的发育模式是在中胚层进入过程中驱动顶端收缩。我们的项目 然后转向系统鉴定 Sbf 和 Rab35 蛋白伴侣在指导棘轮作用中的作用 参与，并检查指导收缩力“转换”行为的细胞信号传导途径 从顶端表面到细胞界面的生成。我们的数据表明，在没有 JAK/STAT 的情况下 发出信号后，Sbf-Rab35 棘轮与胚胎的所有顶端表面接合，从而导致全局 顶端变平和收缩。此外，我们的研究将确定是否有更大的 Upd-JAK-STAT-Pi3K-PIP3-Sbf- Rab35 通路或两条独立通路（PIP3 和 JAK/STAT）已协调调节 棘轮式参与。我们还应用了一种新的基于计算相位的密切圆方法来检测 收缩和扩张位移的活跃期。最后，我们正在开发一种新的线粒体标签异位 重定位测定作为体内募集因子“充分性”的衡量标准，并检查 Akt/mTOR 是否 途径调节细胞棘轮，有可能证明 Akt/mTOR 在 控制上皮细胞拓扑。因此，计划中的项目有可能阐明大量的监管问题 指导上皮组织中细胞棘轮作用的机制层次结构。