Project summary When examined by electron microscopy, neurons can be seen to carry organelles that look like little black dots. These black dots are called dense-core vesicles and they carry many important transmitters that act as neuromodulators, including neuropeptides, nerve growth factors, and monoamines such as dopamine and norepinephrine. Such dense-core vesicle cargos regulate a wide array of behaviors, and defects in such cargos can contribute to numerous mood disorders and other neurological conditions. However, little is understood about the cell biology of how dense-core vesicles are made, acquire cargos and mature, are trafficked to release sites, and ultimately released. Thus, the little black dots in neurons are really a big black box. Adding to the mysteries and complexity of these organelles, many neurons carry multiple dense-core vesicle cargos in the same cell. Are these different cargos copackaged together in the same dense-core vesicles or are they packaged separately in distinct vesicles? The answer to this question is surprisingly known in only a few cases, and the general pattern of copackaging versus segregation of distinct cargos is unclear, but is of key physiological relevance as it determines whether different dense-core vesicle cargos are coreleased or can be released independently. Additionally, in cases where distinct dense-core vesicle subpopulations are known to exist in the same cell, it is unclear how these distinct populations are generated and how cargos are differentially sorted. To begin to address these gaps in understanding, here we aim to establish two model systems for the study of distinct dense-core vesicle populations in the same cell: the ASI sensory neuron in the nematode C. elegans and the rat pancreatic beta-cell line 832/13. Our preliminary data show that members of a known dense-core vesicle biogenesis and maturation pathway, the Rab2/EARP pathway, are required for one subpopulation of dense-core vesicles in both the ASI neuron and the 832/13 cell line, but not for another subpopulation in the same cell. In Aim 1, we will further define the requirements for members of the Rab2/EARP pathway in the biogenesis and maturation of distinct dense-core vesicle subpopulations in the ASI neuron and 832/13 cells. Additionally, we will perform candidate screens in an attempt to identify factors required for the Rab2/EARP-independent generation of dense-core vesicles in both cell types. In Aim 2, we will test the hypothesis that different subpopulations of dense-core vesicles are marked by different isoforms of the synaptotagmin family of calcium sensors. We will also determine whether these synaptotagmins control the release of distinct dense-core vesicle subpopulations. In summary, this project will identify the basic molecules required for the generation and release of distinct subpopulations of dense-core vesicles in the same cell, and set the stage for more mechanistic investigations into how these proc...