Project Summary: Investigating the role of lipid metabolism in the biogenesis of lysosome-related organelles Lysosome-related organelles (LROs) are cell type-specific compartments that share characteristics with ubiquitous conventional lysosomes, yet have specialized functions. LROs are evolutionarily conserved derivatives of the endosomal system that include mammalian melanosomes and platelet dense granules, gut granules in C. elegans, Drosophila pigment granules, and acidocalcisomes in protozoan parasites. Defective LRO biogenesis leads to Hermansky-Pudlak (HPS) and Chediak-Higashi (CHS) syndromes, which cause partial albinism and delayed blood clotting. It is surprising how little attention has been focused on processes controlling LRO size and shape, given how tied LRO morphology is to mechanisms controlling LRO biogenesis, homeostasis, and function. Enlarged LROs are pathogenic in CHS and are diagnostic of HPS1, the most common form of HPS. Prior approaches to identify factors involved in LRO biogenesis have not been designed to identify factors regulating LRO morphology. LRO morphology is controlled by the interplay of membrane trafficking to and from LROs as well as LRO fission and fusion, processes fundamental to the biogenesis, behavior, and activity of LROs. Identifying and characterizing the activity of conserved genes that impact LRO morphology will likely lead to new insights into the processes regulating LRO biogenesis. The goal of this project is to gain a better understanding of the processes controlling LRO morphology by studying acdh-11, which we identified in a screen for mutants that disrupt the morphology of C. elegans LROs called gut granules. ACDH-11 is a highly conserved member of the medically important acyl-CoA dehydrogenase (ACDH) family that catalyze the beta-oxidation and catabolism of fatty acids and amino acids. Our work showing acdh-11(-) mutants cause gut granule enlargement identifies a novel role for an ACDH protein. The FAT-7 fatty acid desaturase becomes expressed and is in part required for LRO enlargement in acdh-11(-) mutants. FAT-7 catalyzes the creation of unsaturated fatty acids, which can promote membrane fluidity by disrupting lipid packing. Our results support the possibility that changing the physical characteristics of membranes influences membrane dynamics to increase gut granule size. The mechanisms by which ACDH- 11 and FAT-7 function to impact LRO size is investigated through four specific aims: (1) investigate how ACDH-11 impacts gut granule size by defining where ACDH-11 acts and whether it functions enzymatically or as a sink for fatty acid substrates; (2) test whether FAT-7 causes the enlargement of gut granules in acdh-11(-) mutants by increasing unsaturated fatty acids within the membranes of intestinal cells; (3) analyze whether gut granule enlargement in acdh-11(-) results from increased membrane fluidity; and (4) identify other genes and pathways acting in acdh-11(-) to promote gut gra...