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)的内稳态和分化 通过典型通路(如Ras/ERK MAPK和PI3K/AKT)调节的信号转导。中断 这些途径通过致病的新生突变导致细胞适合性和父亲年龄相关的变化 导致儿童头面部疾病的SSC突变克隆的积累。其他领域的最新研究 细胞类型表明，信号的动态模式(在几分钟到几小时的范围内)是表型的核心 细胞的行为。然而，到目前为止，用于SSC中的信令要求的数据已经从二进制中导出， 静态措施，这是调和关于……作用的矛盾意见的主要障碍。 SSCs中的特定通路。此外，很难或不可能将上游信号(即增长)联系起来 因子)与特定的下游效应器结合，这是由于平行通路的激活，两者都是在正常的情况下 SSCS和具有致病突变的患者。这项建议的总体目标是开发新的方法 通过激酶转位揭示SSCs中的动态信号模式如何对信息进行编码 报告技术(KTR)，它使定量、实时测量通路活动成为可能。作为证据- 当然，我们将应用这一策略来探测由Canonical驱动的ERK MAPK和PI3K/AKT信号 受体酪氨酸激酶与SSCs的自我更新密切相关，在野生型细胞和那些 驱动父亲年龄相关性颅面部疾病的基因突变。然而，我们期待着我们的 结果将有助于揭示来自细胞外的外部信号如何在多种途径上发挥作用 环境通过网络串扰在内部传输、动态编码和修改。

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.