Control of cell ratcheting engagement during epithelial morphogenesis

项目摘要 上皮组织中的力产生通常是脉冲性的，肌动球蛋白网络产生高张力 细胞皮质在周期性分解之前的收缩力量。细胞骨架力量的这种脉冲性 意味着必须有细胞过程来提取驱动进程的单向变化 单元格形状的变换。在之前的工作中(Juett等人，2017；Miao等人，2019年)，我们发现 细胞骨架力量的产生通过SBF与质膜的内吞重塑相协调。 Rab35管区隔功能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，这个膜细胞是如何 棘轮在特定的细胞表面接合仍不清楚。在建议的研究中，我们会 检查与棘轮相关的信息信号，并将SBF/Rab35隔间行为引导至 收缩界面或细胞尖端，并识别振荡持续时间、幅度、 导致收缩过程的频率和/或方向性。我们的初步数据显示，PIP3 棘轮接洽的关键决定因素-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)在提供基于脂质的膜提示方面的表征 果蝇早期胚胎的形态发生和原肠/棘轮发育动力学在第一个目标中，我们将 也分析质膜超微结构如何通过棘轮过程重塑，并确定 在中胚层侵入期间，PIP水平被发育成驱动心尖收缩的模式。我们的项目 然后系统地鉴定SBF和Rab35蛋白伙伴在指导棘轮调节中的作用 参与，并研究了细胞信号通路，这些信号通路引导着收缩力量的“切换”行为 从根尖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 信号，SBF-Rab35棘轮在胚胎的所有顶面上啮合，导致全局 顶端扁平和收缩。此外，我们的研究将定义更大的UPD-JAK-STAT-PI3K-PIP3-SBF- Rab35途径或两个独立的途径(PIP3和JAK/STAT)是否已被协调调节 棘轮啮合。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 收缩和膨胀位移的活跃期。最后，我们正在开发一种新的mito-tag异位 重新定位试验作为体内募集因子“充分性”的衡量标准，并检查Akt/mTOR 调节细胞棘轮的途径，潜在地展示了Akt/mTOR的一个新的，非常新的功能 控制上皮细胞拓扑结构。因此，计划中的项目有可能阐明一个大型的、监管的 引导上皮组织中细胞棘轮活动的机制的层级。

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.