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

摘要 成年哺乳动物精原干细胞（SSCs）的稳态和分化密切依赖于 通过典型途径调节信号转导（例如，RAS/ERK MAPK和PI3K/AKT）。破坏 这些途径通过致病性从头突变导致细胞适应性的变化， 在儿童中产生颅面疾病的突变SSC克隆的积累。其他领域的最新研究 细胞类型表明，信号的动态模式（在分钟到小时的尺度上）是表型的核心。 细胞的行为。然而，到目前为止，SSC中的信令需求数据一直来自二进制， 静态的措施，是一个主要的障碍，以调和矛盾的意见， SSCs中的特定通路。此外，很难或不可能链接上游信号（即，增长 由于平行途径激活，在正常的背景下， SSC和具有致病性突变的那些。本提案的总体目标是开发新的方法 利用激酶易位，揭示SSC中动态信号模式如何编码信息， 报告基因技术（KTR），它能够定量，实时测量途径的活动。作为证据- 原则上，我们将应用这种策略来探测ERK MAPK和PI 3 K/AKT信号传导， 受体酪氨酸激酶，与SSCs的自我更新密切相关，无论是在野生型细胞和那些 导致父亲年龄相关颅面疾病的基因突变。然而，我们预计， 这些结果将有助于揭示细胞外的外源性信号如何通过多种途径， 环境中的数据在内部传输，动态编码，并通过网络串扰进行修改。

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.