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

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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.