Project Summary Germ cells and somatic cells from an individual carry identical copies of DNA; yet, only germ cells have the potential to give rise to all the cell types of each subsequent generation. A conserved core of germ-granule proteins act as multipotency factors, and they are often repurposed in the soma during development, tissue regeneration, and tumorigenesis. This core consists of Vasa DEAD-box RNA helicases, small RNA binding Argonaute proteins, and LOTUS-Tudor domain proteins that interface and stimulate Vasa and Argonaute activity. The role of these multipotency factors during tumorigenesis and whether they contribute to the phenomenon of cancer/testis (CT) antigen expression in various tumors has not been explored. C. elegans is used to understand how these core proteins are expressed and distributed, what they interact with, and their role in fertility and developmental plasticity. The specific aims in the parent grant look at 1) the role of germ granules in protein expression and turnover, primarily focusing on the novel interaction of GLH/Vasa with PCI scaffolding complexes, 2) how GLH promotes the differential translation of spermatogenic transcripts, and 3) how GLH and a new LOTUS-Tudor protein called LOTR-1 cooperates to maintain germline integrity. Findings from this project reveal novel ways to manipulate cellular pluripotency and expose the potential therapeutic targets that directly regulate the cytoplasm instead of nuclear gene expression networks. In the parent grant, two microscopy-based assays have been refined to 1) detect protein proximity interactions with core germ-granule proteins and 2) determine how these proteins impact differential translation. These microscopy assays rely on an epifluorescent microscope equipped with 3D deconvolution software - a system that is routinely used 20-40 hours each week. This microscope was acquired nine years ago with start-up funds provided to the PI, but it requires drivers and an operating system no longer available or supported. The administrative supplement replaces the aging imaging system with the modern equivalent, allowing the transfer of some hardware from the old to the new system. Acquisition speed, resolution, and deconvolution capacity are improved by orders of magnitude with this equipment upgrade. Replacing this equipment ensures the continuity, output, and quality of data collected for experiments outlined in the parent grant.