Timing live cell cycle length in diverse tissues

测定不同组织中活细胞周期长度

• 批准号: 10195312
• 负责人: Shangqin Guo
• 金额: $ 20.94万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195312
• 关键词: Acceleration; Adult; Biological Models; Cell Cycle; Cell Differentiation process; Cell division; Cell model; Cells; Chimeric Proteins; Color; Communities; Coupled; Development; Disease; Erythrocytes; Erythroid; Flow Cytometry; Fluorescence; Generations; Half-Life; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneity; Histone H2B; Homeostasis; Hour; Image; In Situ; Individual; Injury; Kidney; Kidney Diseases; Kinetics; Label; Length; Life; Mammalian Cell; Measures; Mediating; Megakaryocytes; Mitosis; Mitotic; Mouse Strains; Myelogenous; Myelopoiesis; Myeloproliferation; National Institute of Diabetes and Digestive and Kidney Diseases; Performance; Physiologic pulse; Population; Population Sizes; Proteins; Proteome; Regulation; Reporter; Reporting; Research; S Phase; Solid; Speed; Standardization; Testing; Time; Tissues; Urologic Diseases; Variant; Work; cell determination; cell type; design; experience; granulocyte-monocyte progenitors; hematopoietic stem cell quiescence; hematopoietic tissue; in vivo; mathematical model; progenitor; ratiometric; response; sample fixation; stem; success; technology development; time interval; tool; tool development

Timing live cell cycle length in diverse tissues Abstract Cellular dynamics underly tissue homeostasis and its abnormality defines many disease states. Timing how quickly cells form tissues requires knowing the cellular generational time, or cell cycle speed, most conveniently defined as the time interval between two consecutive mitoses. Determination of cell cycle speed for cells in deep tissues has relied on S-phase activity to incorporate pulsed labels, or cell division mediated- dilution of saturated labels. Thus, cell cycle rates have only been calculatable as a population average upon fixation, or after the label has been diluted to a certain level within the detectible range, among other limitations. An instantaneous readout of cell cycle speed in individual live cells that enable their isolation has been out of reach. In response to the call of Catalytic Tool and Technology Development in Kidney, Urologic, and Hematologic Diseases, I propose to develop new genetically encoded live cell cycle speed reporter. Mouse strain expressing the new reporter will be established for detecting a wide range of cell cycle speed by fluorescence, in situ or by flow cytometry, to catalyze the research on cellular dynamics in diverse tissues. We will build on our recent success in developing a first-of-its-kind live cell cycle speed reporter by exploiting the differential half-life of a color changing protein, the fluorescent timer (FT). We chose the kinetic variant that emits blue fluorescence when newly synthesized for ~1.2 hours, before converting into a red protein permanently during maturation. Expressed as a fusion protein to core histone H2B, cell cycle length of individual cells can be determined by the ratio between the two fluorescence: the faster the cell cycle, the bluer a cell appears. While this first reporter demonstrated the proof-of-principle and exceptional performance in resolving short cell cycles, such as those of the erythroid progenitors and myeloid-committed progenitors, the vast cell types dividing at slower rates were not resolvable. Through this proposal, we will test new design features so that the live cell cycle speed relevant for most mammalian cell types in vivo can be conveniently determined with a single genetically encoded reporter construct, H2B-FTmHaloD2. Given the naturally existing wide range of cell cycle speed in the hematopoietic tissues and our own expertise in studying it, we will use the hematopoietic stem and progenitor cells at baseline and during injury as model cell types to test, calibrate and standardize workflows for how a new ratiometric reporter can be used to determine cellular dynamics and sort for live cells from diverse tissues. The new design features of the H2B-FTmHaloD2 should also readily resolve the slow cell cycles present in solid tissues such as the kidney.

不同组织中活细胞周期长度的计时 摘要 细胞动力学是组织稳态的基础，其异常定义了许多疾病状态。定时 细胞形成组织的速度需要知道细胞的世代时间，或细胞周期的速度， 方便地定义为两个连续有丝分裂之间的时间间隔。细胞周期速度的测定 对于深层组织中的细胞，依赖于S期活性来掺入脉冲标记，或细胞分裂介导的- 饱和标记的稀释。因此，细胞周期率只能作为群体平均值计算， 在其它限制中，标记物可以在固定之后或在标记物已被稀释到可检测范围内的某一水平之后被标记。 能够分离单个活细胞的细胞周期速度的瞬时读数已经超出了 到达。为响应肾脏、泌尿外科和泌尿外科催化工具和技术开发的号召， 血液病，我建议开发新的基因编码的活细胞周期速度报告。鼠标 将建立表达新报告基因的菌株，用于检测宽范围的细胞周期速度， 荧光，原位或通过流式细胞术，以催化对不同组织中细胞动力学的研究。 我们将建立在我们最近成功开发的第一个同类活细胞周期速度报告， 利用变色蛋白质的不同半衰期，荧光计时器（FT）。我们选择了动力学 新合成时发出蓝色荧光的变体约1.2小时，然后转化为红色 蛋白质在成熟过程中的作用。表达为核心组蛋白H2 B的融合蛋白，细胞周期长度为 单个细胞可以通过两种荧光之间的比率来确定：细胞周期越快， 一个细胞出现了。虽然这第一个记者展示了原理证明和卓越的性能， 解决短细胞周期，如红系祖细胞和髓系定向祖细胞的细胞周期， 以较慢速率分裂的大量细胞类型是不可分辨的。通过这项提案，我们将测试新的设计 特征，使得可以方便地测量与体内大多数哺乳动物细胞类型相关的活细胞周期速度。 用单个遗传编码的报告构建体H2 B-FTmHaloD 2测定。鉴于自然存在的 广泛的细胞周期速度在造血组织和我们自己的专业知识，在研究它，我们将使用 在基线和损伤期间作为模型细胞类型的造血干细胞和祖细胞，以测试、校准和 标准化工作流程，用于如何使用新比率报告器来确定细胞动力学和分类 从不同组织中提取活细胞H2 B-FTmHaloD 2的新设计功能也应该很容易解决 存在于实体组织如肾脏中的缓慢细胞周期。