The endoplasmic reticulum (ER) is responsible for the synthesis of a variety of membrane proteins and lipids, which are delivered to their destination by vesicular transport; however the ER also forms direct contacts with the plasma membrane (PM). Constitutive ER-PM (cortical ER) contacts have been shown to be crucial in muscle for propagating calcium signals and inducible contacts in all cells (mediated by Stim1-Orai) are responsible for store-operated Ca2+ entry. Recently our laboratory characterized a new type of ER-PM contact mediated by a set of ER proteins, the Extended-Synaptotagmins (Esyt1-3). These form contacts with the PI(4,5)P2 in PM via their C2 domains and are additionally regulated by increase in cytosolic Ca2+. The crystal structure of E-Syts shows that they contain an SMP domain, a tubular module with a hydrophobic cavity that harbors lipids. This is consistent with growing evidence for a role of membrane contact sites in lipid regulation and transfer between the ER and PM. E-Syts are likely to be important in neurons as they are highly expressed in the brain. Since neuron processes are often very long, vesicular transport of newly synthesized membrane lipids via the secretory pathway is difficult. Direct transfer of newly synthesized via E-Syt contact sites could be especially important during outgrowth of long axons and dendrites or during neurotransmission when rapid depletion of membrane proteins requires efficient replenishment from the ER. Indeed, neurons are particularly sensitive to disruptions in lipid transfer, which can cause both membrane trafficking defects and impaired neurite outgrowth. The goal of this proposal is to test the hypothesis that E-Syt mediated contacts play a critical role in neuron maintenance, synaptic transmission, and neurite development. I will characterize E-Syt mediated ER-PM contacts in neurons by examining cortical ER in the soma, axons and dendrites. I will use serial blockface scanning electron microscopy, TIRF, spinning disc confocal, and in vivo two-photon microscopy to examine these contacts in brain tissue, cultured neurons, and living animals. I will test the hypothesis that E-Syt contacts are involved i neurotransmission by determining the effect of depolarization on E-Syt recruitment to the membrane. Furthermore I will determine whether E-Syt contacts play a role in neurite development by assessing the effect of E-Syt knockdown on branching and dendritic spine development.