Mechanisms governing stem cell coordination by the niche

通过生态位控制干细胞协调的机制

• 批准号: 10389225
• 负责人: Kari Lenhart
• 金额: $ 10万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389225
• 关键词: 3-Dimensional; Administrative Supplement; Biological Models; Cell Communication; Cell Lineage; Cell physiology; Complex; Cytokinesis; Data; Daughter; Drosophila genus; Ensure; Environment; Excision; F-Actin; Funding; Genetic; Germ Cells; Goals; Hair follicle structure; Hematopoietic; Homeostasis; Hour; Image; Laser Scanning Confocal Microscopy; Lasers; Lateral; Maintenance; Microscope; Modification; Molecular; Molecular Genetics; Outcome; Output; Ovary; Population; Production; Productivity; Resolution; Scanning; Signal Transduction; Source; Structure; System; Testis; Time; Tissues; Tumor Stem Cells; Work; adult stem cell; cell type; daughter cell; detector; genetic manipulation; germline stem cells; imaging capabilities; improved; insight; instrumentation; parent grant; prevent; programs; quantum; reconstruction; spatiotemporal; sperm cell; stem cell population; stem cell proliferation; stem cells

Project Summary / Abstract Coordination between adult stem cells is essential to maintain tissue homeostasis and prevent tumorous overgrowth. Many structures, including the hair follicle, hematopoietic network and developing ovary require tight control over stem cell proliferation and coordination of daughter cell production from distinct stem cell lineages. In most cases, the molecular mechanisms orchestrating this coordination are largely unknown. Leveraging the power of Drosophila genetics and establishing a system for longitudinal (20+ hours) live imaging of stem cells within an endogenous niche we have begun to reveal the mechanisms controlling stem cell coordination in the testis. Somatic stem cells and germline stem cells (GSCs) of the testis must generate daughters in a precise 2:1 ratio for germ cells to effectively differentiate into sperm. Our live imaging has revealed a modified cytokinesis program in GSCs as the mechanism to coordinate release of one GSC daughter only after it correctly associates with two daughters of the somatic stem cell lineage. This modified cytokinesis program is controlled at two stages—a pause regulated by Jak/STAT signaling from the niche and a trigger for completion of cytokinesis derived from the somatic stem cells. Both control points must be properly executed or stem cell cytokinesis fails, stem cell tumors form and germ cells fail to differentiate. While we have identified the source of both the pause and trigger, the mechanisms by which these signals control GSC cytokinesis remain unknown. In the parent grant, we propose to interrogate the specific mechanisms by which niche signals and somatic stem cells combine to regulate GSC cytokinesis using molecular genetics and extended live imaging. Here, we request an administrative supplement for the acquisition of fast and super-resolution laser scanning confocal microscopy, which will improve the imaging capabilities, quality and data output (productivity) of our R01-funded projects. The new instrumentation will enable imaging of fixed and live testes faster and with less photodamage as well as unprecedented resolution (100 nm lateral, 200 nm axial). This enhanced resolution will aid in identification of altered F-actin structure at the intercellular bridge between GSC-daughter pairs investigated in Aim1 of the parent grant. In our studies of temporal dynamics in abscission machinery localization to the GSC-daughter intercellular bridge (Aim2 of parent grant), the new instrumentation will enable tracking of ESCRT machinery with unprecedented spatiotemporal resolution in 2D and 3D using detectors with superior quantum efficiency. Through the use of advanced modulations such as dynamic enhancement and adaptive image quality determination and reconstruction, we will be able to acquire high-quality time-lapse data with improved resolution. This is in conjunction with the ability to acquire images from multiple (up to three) wavelengths simultaneously—an essential feature for our work dissecting the complex interactions between somatic and germline stem cells through live imaging of both populations (Aim3 of parent grant). Outcomes will shed valuable insight into niche-regulated cytokinesis modifications in stem cells. In addition, this work will provide the first real-time, high-resolution analysis of stem cell interactions within an endogenous niche that are essential for maintenance of tissue homeostasis.

项目概要/摘要 成体干细胞之间的协调对于维持组织稳态和预防 肿瘤过度生长。许多结构，包括毛囊、造血网络和 发育中的卵巢需要严格控制干细胞增殖和子细胞的协调 来自不同干细胞谱系的细胞生产。在大多数情况下，分子机制 编排这种协调在很大程度上是未知的。利用果蝇的力量 遗传学并建立一个系统，用于对体内干细胞进行纵向（20+小时）实时成像 内源性利基我们已经开始揭示控制干细胞的机制 睾丸的协调。睾丸的成体干细胞和生殖系干细胞 (GSC) 必须 以精确的 2:1 比例生成女儿，使生殖细胞有效分化为精子。 我们的实时成像揭示了 GSC 中修改的胞质分裂程序，作为 只有在正确关联了一个 GSC 子代的两个子代后，才协调释放它 成体干细胞谱系。这种修改后的胞质分裂程序分两个阶段进行控制——a 暂停由来自利基的 Jak/STAT 信号调节，并触发完成 源自成体干细胞的胞质分裂。两个控制点都必须正确执行 或者干细胞胞质分裂失败，干细胞肿瘤形成并且生殖细胞无法分化。当我们 已经确定了暂停和触发的来源，以及这些暂停和触发的机制 控制 GSC 胞质分裂的信号仍不清楚。在家长补助金中，我们建议询问 生态位信号和成体干细胞结合调节的具体机制 使用分子遗传学和扩展实时成像进行 GSC 胞质分裂。在此，我们要求 获取快速超分辨率激光扫描的行政补充 共焦显微镜，这将提高成像能力、质量和数据输出 我们 R01 资助项目的（生产力）。新仪器将能够对固定的图像进行成像 和活睾丸更快，光损伤更少，以及前所未有的分辨率（100 nm 横向，200 nm 轴向）。这种增强的分辨率将有助于识别改变的 F-肌动蛋白 在 Aim1 中研究的 GSC-子对之间的细胞间桥结构 家长补助金。在我们对离离机制的时间动力学的研究中，局部化到 GSC-子细胞间桥（父资助的目标2），新仪器将能够 使用 2D 和 3D 方式以前所未有的时空分辨率跟踪 ESCRT 机械 具有卓越量子效率的探测器。通过使用先进的调制，例如 动态增强和自适应图像质量确定和重建，我们将 能够以更高的分辨率获取高质量的延时数据。这与 同时从多个（最多三个）波长获取图像的能力 我们工作的基本特征是剖析体细胞和种系之间复杂的相互作用 通过对两个群体的实时成像来检测干细胞（父母资助的目标3）。结果将会脱落 对干细胞中生态位调节的胞质分裂修饰的宝贵见解。此外，这 这项工作将首次对干细胞内的相互作用进行实时、高分辨率的分析。 内源性生态位对于维持组织稳态至关重要。