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In mammals, activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery. However, the biochemical evidence for this balance and coordination is largely lacking. To investigate this we initially used acute metabolic radiolabeling of stimulated primary mouse neurons to follow the fate of polypeptides being newly synthesized. We observed polypeptides being newly translated exclusively during neuronal stimulation were rapidly degraded by the neuronal membrane proteasome (NMP) and not the cytosolic proteasome. This turnover correlated with enhanced production of NMP-derived peptides into the extracellular space which have the capacity to mediate neuronal signaling in part through NMDA receptors. Using in-depth, global, and unbiased mass spectrometry, we identified the nascent protein substrates of the NMP. Among these substrates, we found that immediate-early gene products c-Fos and Npas4 were targeted to the NMP during ongoing activity-dependent protein synthesis. Moreover, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP. This generates a new modality of neuronal signaling in the form of extracellular peptides with potential significance for our understanding of nervous system development and function.

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Exocytosis occurs in all living cells and is essential for many cellular processes including metabolism, signaling, and trafficking. During exocytosis, cargo loaded vesicles dock and fuse with the plasma membrane to release their content. To accommodate different cargos and cellular needs exocytosis must occur across scales; From synaptic vesicles that are only ~50nm in diameter, and neuroendocrine vesicles that are in the ~500nm range to giant secretory vesicles filled with viscous cargo, such as in the acinar cells in the exocrine pancreas, that reach up to a few µm in diameter. Yet, how fusion and content release are adapted to remain function across these scales is not well understood. It is well established that during exocytosis of small vesicles, vesicle fusion can proceed through one of two pathways: The first is complete incorporation, when the vesicular membrane fuses to the target membrane and the fusion pore expand irreversibly, incorporating the vesicular membrane into the target membrane. The second is “kiss-and-run”, when the fusion pore flickers, opening briefly and collapsing rapidly into two separate membranes. I am interested in understanding how exocytosis occurs in giant vesicles witch challenge efficient secretion and membrane homeostasis due to their massive size and viscous content. I am using the salivary gland of D. Melanogaster, as a model system for giant vesicles secretion. The vesicles in the gland measure between 5-8 µm, fuse and secrete viscous content into a preformed lumen. To visualize the secretion process, I adapted a method for super-resolution microscopy to live-gland imaging. I observed that fusion pores of giant vesicles expand to a stable opening of up to 3µm and slowly constricts down to hundreds of nm or less during secretion. Because constricting a membrane pore from “infinity” in molecular terms, back to a very narrow ‘stalk’ demands an investment of energy, I hypothesized that this is mediated by a specialized protein machinery. I am currently screening for the components of the machinery using the enormous power of Drosophila genetics by taking a candidate gene approach. My preliminary results identify the BAR domain containing protein, MIM (missing in metastasis) as a key regulator of pore dynamics, leading to new and exciting insights into the molecular mechanism of cellular secretion and membrane homeostasis in live tissues.

Interferons (IFNs) were the first cytokines discovered over half a century ago as agents that interfere with viral infection. IFNs have been established as pleiotropic, multifunctional proteins in the early immune response. They exhibit antiviral and antiproliferative effects, in addition to various immunomodulatory activities. Human type I IFN family consists of 16 members, all acting through the same cell surface receptors, IFNAR1 and IFNAR2. Here, we show that synthetic interferon receptors can activate the Jak/Stat pathway using non-physiological ligands. High affinity GFP and mCherry nanobodies were fused to transmembrane and intracellular domains of the receptors in attempt to perform in-vivo and in-vitro biophysical assays. This will help in better understanding the structure - function relationship of the receptors and their associated ligands.