Methods for detection of dynamic intracellular signals in single adult spermatogonial stem cells

单个成体精原干细胞动态细胞内信号的检测方法

基本信息

批准号：
10666116
负责人：
Todd R Evans
金额：
$ 25.25万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10666116
关键词：
Address Adult Aging Alleles Apert syndrome Behavior Biochemical Cell Culture System Cell Nucleus Cell Surface Receptors Cells Chemicals Child Clone Cells Cytoplasm Data Detection Disease Engineering Environment FGFR2 gene Fibroblast Growth Factor Fibroblast Growth Factor Receptors Gene Abnormality Genes Genetic Genotype Germ Cells Goals Growth Factor Homeostasis Hour Link Literature MAP Kinase Gene MEKs Mammals Maps Measurement Measures Mediating Methodological Studies Methodology Methods Modality Monitor Mus Mutation Noonan Syndrome Output PI3K/AKT PTPN11 gene Paternal Age Pathogenesis Pathogenicity Pathway interactions Pattern Phenotype Phosphotransferases Population Population Study Process Production Proliferating Ras/Raf Receptor Protein-Tyrosine Kinases Reporter Role Signal Pathway Signal Transduction Somatic Cell Techniques Technology Testis Time Variant Western Blotting age related behavioral phenotyping cell behavior cell type craniofacial disorder de novo mutation detection method enzyme substrate experimental study extracellular fitness fluorophore gain of function mutation gene product glial cell-line derived neurotrophic factor improved mutant novel offspring pharmacologic phase change receptor response self-renewal sperm cell stem cells technology platform temporal measurement transmission process

项目摘要

Abstract Homeostasis and differentiation of adult mammalian spermatogonial stem cells (SSCs) depend on tightly regulated signal transduction through canonical pathways (e.g., RAS/ERK MAPK and PI3K/AKT). Disruption of these pathways by pathogenic de novo mutations leads to changes in cell fitness and paternal age-related accumulation of mutant SSC clones that generate craniofacial disorders in children. Recent studies in other cell types show that dynamic patterns of signaling (on the scale of minutes to hours) are central to phenotypic behavior of cells. Yet, the data for signaling requirements in SSCs until now have been derived from binary, static measures, representing a major barrier to reconciling paradoxical observations regarding the roles of specific pathways in SSCs. Furthermore, it has been difficult or impossible to link upstream signals (i.e., growth factors) with specific downstream effectors due to parallel pathway activation, both in the context of normal SSCs and those with pathogenic mutations. The general goal of this proposal is to develop novel methodology to reveal how information is encoded by dynamic signaling patterns in SSCs, using kinase translocation reporter technology (KTR), which enables quantitative, real-time measurement of pathway activity. As proof- of-principle, we will apply this strategy to probe ERK MAPK and PI3K/AKT signaling driven by canonical receptor tyrosine kinases that are closely linked to self-renewal of SSCs, both in wild type cells and those with mutations in genes that drive paternal age-associated craniofacial disorders. However, we anticipate our results will be useful across a variety of pathways for revealing how extrinsic signals from the extracellular environment are transmitted internally, dynamically encoded, and modified by network cross talk.

抽象的成年哺乳动物精原干细胞（SSC）的稳态和分化紧密依赖于通过经典途径（例如 RAS/ERK MAPK 和 PI3K/AKT）调节信号转导。扰乱这些途径通过致病性从头突变导致细胞适应性和父亲年龄相关的变化突变型 SSC 克隆的积累会导致儿童颅面疾病。最近在其他方面的研究细胞类型表明信号的动态模式（以分钟到小时为单位）是表型的核心细胞的行为。然而，到目前为止，SSC 中的信令要求数据都是从二进制导出的，静态措施，是调和有关角色角色的矛盾观察的主要障碍 SSC 中的特定途径。此外，连接上游信号（即增长信号）一直很困难或不可能。由于并行途径激活，在正常情况下具有特定的下游效应器 SSC 和具有致病性突变的 SSC。该提案的总体目标是开发新颖的方法利用激酶易位揭示 SSC 中动态信号传导模式如何编码信息报告技术 (KTR)，能够定量、实时测量通路活性。作为证明—— 原则上，我们将应用这种策略来探测由规范驱动的 ERK MAPK 和 PI3K/AKT 信号传导受体酪氨酸激酶与 SSC 的自我更新密切相关，无论是在野生型细胞还是在野生型细胞中导致父亲年龄相关颅面疾病的基因突变。然而，我们预计我们的结果将有助于跨多种途径揭示来自细胞外的外在信号如何环境在内部传输，动态编码，并通过网络串扰进行修改。