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: 10544507
• 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/10544507
• 关键词: 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; Cytoskeleton; Data; Data Analyses; Development; Dimensions; Drosophila genome; Drosophila genus; Embryo; Epithelial Cells; Epithelium; FRAP1 gene; Frequencies; Generations; Goals; Image; Individual; Intention; Intercalated Cell; Lipids; Mass Spectrum Analysis; Measurement; Measures; Membrane; Mesoderm; Mitochondria; Morphogenesis; Morphology; Nature; PH Domain; PIK3CG gene; Pathway interactions; Pattern; Periodicals; 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; cell cortex; cell motility; constriction; course development; gastrulation; in vivo; insight; 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.

项目摘要 上皮组织中的力通常是脉动的，肌动球蛋白网络产生高张力 循环拆卸之前，细胞皮质的收缩力。细胞骨架力的这种脉冲性质 意味着必须有蜂窝过程才能提取单向变化以驱动过程 细胞形状的转换。在先前的工作中（Jewett等，2017； Miao等，2019），我们发现 细胞骨架力的产生与质膜通过SBF-的内吞重塑协调 RAB35管状室室功能以稳定收缩的细胞表面并允许细胞收缩 顶点（顶部收缩）或细胞界面（细胞插入）。但是，这种膜细胞如何 棘轮在特定的细胞表面上参与了尚不清楚。在拟议的研究中，我们将 检查参与赛车并将SBF/RAB35隔间行为的信息信号 收缩界面或细胞顶点，并确定振荡持续时间，放大器的基本变化 频率和/或导致收缩加工性的方向性。我们的初步数据表明PIP3是 赛车参与的关键决定者 - 通过我们拟议的工作，我们将执行第一个 磷脂酰肌醇磷酸盐（PIP）的表征在提供基于脂质的膜提示的情况下 早期的果蝇胚胎中的形态发生和过口术/棘轮动力学。在第一个目标中，我们将 还分析赛车过程如何重塑质膜超微结构并确定是否是否 在中胚层浸入期间，开发了PIP水平以驱动顶端收缩。我们的项目 然后移动到指导赛车的SBF和RAB35蛋白伴侣的系统鉴定 参与，并检查指导收缩力“切换”行为的细胞信号通路 从顶部表面到细胞界面的产生。我们的数据表明，在没有JAK/STAT的情况下 信号传导，SBF-RAB35棘轮在胚胎中的所有顶部表面都参与 顶部的扁平和收缩。此外，我们的研究将定义较大的UPD-JAK-STAT-PI3K-PIP3-SBF- RAB35途径或两个独立途径（PIP3和JAK/STAT）已协调以调节 赛车参与。我们还采用新的基于计算阶段的示波圆方法来检测 积极的收缩和膨胀位移。最后，我们正在开发一个新的mito-tag ecopic 重新定位测定作为体内募集因素的“充分性”的量度，并检查AKT/MTOR是否是 途径调节细胞赛车，有可能证明Akt/mTOR的新型，高度新颖的功能 控制上皮细胞拓扑。那就是计划的项目有可能阐明大型监管 指导细胞竞争在上皮组织中的机制的层次结构。