During the formation of the female gamete the cell cycle events of meiosis must be precisely coordinated with the ongoing differentiation of the oocyte. We use Drosophila melanogaster as a model system to define the pathways that regulate early meiotic progression and oocyte development. To achieve this goal we use forward genetic, biochemical and cell biological approaches. In single celled eukaryotes, the pathways that monitor nutrient availability are central to regulating the meiotic program and spore development. However, how metabolic inputs influence meiotic progression and gametogenesis remains poorly understood in metazoans. Our current studies focus on understanding how metabolism influences meiotic progression and oocyte development. Target of rapamycin complex 1 (TORC1) is a master regulator of metabolism in eukaryotes that integrates information from multiple upstream signaling pathways. Over the last year we have determined that, as is observed in yeast, the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. We find that depleting Iml1, the catalytic component of the GATOR1 complex, in the female germline using an RNAi based methodology delays the mitotic/meiotic transition. Conversely, mutations in the nutrient sensitive kinase Tor result in premature meiotic entry. Thus in Drosophila females, high TORC1 activity inhibits the mitotic/meiotic transition and gamete development while low TORC1 activity promotes these events. Currently, we are using genetic strategies to determine what downstream targets of TORC1 mediate the meiotic delay. Additionally, we are examining if the GATOR1 complex has critical functions in latter stages of oogenesis. Towards this end we have generated deletion alleles, using the CRISPR/CAS9 system, of the genes that encode the GATOR1 components Nitrogen permease regulators 2 and 3 (Nprl2 and Nprl3).In yeast Npr2 and Npr3 mediate an essential response to amino acid limitation upstream of TORC1. In mammals, Nprl2 is a putative tumor suppressor gene that inhibits cell growth and enhances sensitivity to numerous anticancer drugs including cisplatin. However, the precise role of Nprl2 and Nprl3 in the regulation of metabolism in metazoans remained poorly defined. Over the last year we have shown that that the central importance of Nprl2 and Nprl3 in the response to amino acid starvation has been conserved from single cell to multicellular animals. We find that in Drosophila, Nprl2 and Nprl3 physically interact and are targeted to lysosomes and autolysosomes. Using oogenesis as a model system, we showed that Nprl2 and Nprl3 inhibit TORC1 signaling in the female germline in response to amino acid starvation. Moreover, the inhibition TORC1 by Nprl2/3 is critical to the preservation of female fertility during times of protein scarcity. In young egg chambers the failure to down-regulate TORC1 in response to amino acid limitation triggers apoptosis. Thus, our data suggest the presence of a metabolic checkpoint that initiates a cell death program when TORC1 activity remains inappropriately high during periods of amino acid and/or nutrient scarcity in oogenesis. Finally, we demonstrated that Nprl2/3 work in concert with the TORC1 inhibitors Tsc1/2 to fine tune TORC1 activity during oogenesis and that Tsc1 is a critical downstream effector of Akt1 in the female germline.
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