Browsing by Author "Sangari, Sadaf"

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    Studying the Interaction of RACK-1 and GLD-1 in the Germline of Caenorhabditis elegans
    (2022-08-18) Sangari, Sadaf; Hansen, Dave; Cobb, John; Samuel, Marcus; Childs, Sarah
    Stem cells are undifferentiated cells that can proliferate to produce more stem cells or differentiate to produce specialized cells. Two gonad arms are present within the germline of the hermaphrodite C. elegans, where proliferating germline stem cells (GSCs) are located at the distal end while meiotic cells occur more proximally, eventually differentiating as gametes. The balance between proliferation and differentiation allows this spatial patterning to be maintained in the germline. Our lab has been studying the influence of highly conserved scaffold protein RACK-1 (Receptor for Activated C Kinase 1) and translational repressor GLD-1 (Defective in Germline Development 1) on the proliferation differentiation decision of GSCs. RACK-1 is involved in ribosome assembly and the miRISC pathway. Meanwhile, GLD-1 functions in germline stem cell entry and progression through meiosis as well as oogenesis and sex determination. Previous work had shown that an absence of rack-1 results in lower GLD-1 levels as well as GLD-1 mis-localization to P granules. Furthermore, an absence of rack-1 resulted in an over-proliferation phenotype in a glp-1(ar202gf) background. As a result, a model was proposed where a potential interaction between RACK-1 and GLD-1 was predicted. In this thesis, I investigated the possible physical interaction between RACK-1 and GLD-1. RACK-1 strains were tagged using CRISPR technology. These strains were shown to behave similarly to a wildtype organism through immunostaining and brood size assay experiments. As a result, the strains were used as reagents for co-immunoprecipitation experiments to detect an interaction between GLD-1 and RACK-1. The same CRISPR tagged strains were used to show that RACK-1 was expressed in the cytoplasm and on cellular membranes in the germline. Several variations of co-immunoprecipitation experiments were performed, where an interaction between RACK-1 and GLD-1 was detected when GLD-1 was immunoprecipitated. The pulldown of RACK-1 with GLD-1 indicated a physical interaction within the cytoplasm that may be direct or indirect. This research gave us insight into the potential influence of this interaction on the proliferation differentiation decision of GSCs. RACK-1 may help GLD-1 by stabilizing it, in turn allowing it to function normally as a translational repressor, which allows for an organized germline.