The long-term goals of this proposal are to define the cellular and molecular determinants of organismal heme homeostasis. Heme, an iron-containing organic ring, functions as a vital cofactor responsible for diverse biological functions, and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. The current paradigm states that cellular requirements for heme are fulfilled by the cell’s internal capacity to regulate and synthesize its own heme. Our paradigm-shifting hypothesis is that cellular heme levels are not only maintained by internal heme synthesis (cell-autonomous), but also by distally located proteins which signal systemic heme requirements to an inter-organ heme trafficking network (cell- nonautonomous). Although emerging evidence support the existence of a systemic cell-nonautonomous heme communication system in mammals, this concept has remained unexplored. Caenorhabditis elegans is a unique animal model to determine if such systemic signaling pathways exist as C. elegans allows systemic heme homeostasis to be manipulated nutritionally and genetically, and their optical transparency allows for in vivo monitoring of heme signals between tissues at subcellular resolution. In the last funding period, we identified a novel bidirectional signaling pathway of heme status between the intestine and neuron. We showed that distal organs have dedicated mechanisms to communicate and coordinate their heme status with the crucial heme storage and transport organ – the intestine via HRG-7 (cathepsin E), DBL-1 (BMP5), SMA-9 (SHN) and HRG-1 (SLC48A1). In this proposal, we will test the hypothesis that inter-organ heme communication is coordinated by both, HRG-7 dependent and HRG-7 independent pathways. This hypothesis is supported by exciting new preliminary data from proteomic, genome-wide RNAi, forward genetic, and RNAseq studies. We will (a) elucidate the molecular mechanisms for a secreted EGF-domain protein in directly regulating HRG-7-dependent signaling pathway; (b) assess the molecular requirement for a transmembrane potassium/ion channel in regulating HRG-7-independent inter-tissue heme signaling pathway; and (c) uncover the mechanisms for adaptor protein complexes in coordinating intestinal heme transport with extra-intestinal heme allocation to regulate systemic heme signaling. Our goals are to acquire a deep understanding of heme-dependent signaling at a tissue and subcellular resolution within the context of an intact animal, as 95% of the total body iron quota in humans is within heme proteins.