2009 — 2012 |
Sirkis, Daniel W. |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
Biogenesis of the Regulated Secretory Pathway @ University of California, San Francisco
DESCRIPTION (provided by applicant): Neuromodulators including monoamine neurotransmitters and neural peptides control a wide range of behavioral phenomena, and their dysregulation contributes to neuropsychiatric disease, but we still understand little about the cellular mechanisms that underlie their regulated release. In particular, we do not understand how proteins sort to the large dense core vesicles (LDCVs) that mediate regulated release, rather than to the vesicles that mediate constitutive secretion. Previous work has even suggested a role for lumenal and membrane rather than cytoplasmic interactions of the kind that contribute to other trafficking events such as endocytosis. To identify factors involved in the formation of LDCVs, I am using the vesicular monoamine transporter (VMAT2), a polytopic membrane protein which fills secretory vesicles with monoamine neurotransmitters for exocytotic release. VMAT2 sorts efficiently and directly into the regulated secretory pathway (RSP), and mutations in a conserved, cytoplasmic dileucine-like motif divert VMAT2 to the constitutive pathway, increasing cell surface delivery. This motif appears important for sorting VMAT2 directly into LDCVs, suggesting that, in contrast to previous work, cytoplasmic factors mediate the sorting of VMAT2to the RSP and thus contribute to the biogenesis of LDCVs. We aim to identify the cellular machinery involved in formation of the RSP, using an unbiased RNAi screen in Drosophila S2 cells. Further, we will characterize the role of identified cellular factors in mammalian PC12 cells. In particular, we will determine whether these factors are responsible specifically for the sorting of VMAT2,or more generally for biogenesis of the pathway. Our studies will enable us to identify cellular factors involved in formation of the regulated secretory pathway. This information will allow us to manipulate regulated and constitutive secretion and determine their relative role in normal behavior and psychiatric illness
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1 |
2015 — 2017 |
Sirkis, Daniel W. |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Biochemical Dissection of Amyloid Precursor Protein Transport From the Golgi @ University of California Berkeley
? DESCRIPTION (provided by applicant): This is a proposal for an F32 award for Dr. Daniel Sirkis, a postdoctoral associate in the Department of Molecular and Cell Biology at the University of California, Berkeley. Dr. Sirkis is a cell biologist focusing on the intracellular trafficking of proteins implicated in neurodegenerative disease. This F32 will provide Dr. Sirkis with the support required to accomplish the following goals: (i) to gain experience in the biochemical reconstitution of vesicle trafficking events; (ii) to learn state-of-the-art genome-editing techniques, such as CRISPR/Cas9-mediated mutagenesis; (iii) to use these techniques to clarify the relationship between the intracellular sorting of the amyloid precursor protein (APP and the generation of its proteolytic cleavage product, amyloid-ß (Aß), both of which have been implicated in Alzheimer's disease (AD); and (iv) to gain additional experience in scientific communication and mentoring that will be required for a smooth transition to an independent research career. Dr. Sirkis has chosen to carry out his postdoctoral studies under the sponsorship of Dr. Randy Schekman, a cell biologist with deep expertise in vesicle trafficking and biochemical reconstitution. The proposed research project focuses on the intracellular sorting of APP at the trans-Golgi network (TGN). Because the TGN has been implicated as a major site of Aß generation, this work has the potential to shed light on pathogenic processes involved in AD. In Aim 1, Dr. Sirkis will determine whether small GTP- binding proteins found to influence Aß production are involved in APP trafficking at the TGN. In Aim 2, Dr. Sirkis will determine whether the transmembrane protein SorLA, a known risk factor for late-onset AD, influences Aß production by facilitating APP transport from either the TGN or endosomes. In Aim 3, Dr. Sirkis will determine whether novel AD risk factor phospholipase D3 influences Aß production via the endoplasmic reticulum or the TGN. The overarching goals of this project are (i) to characterize how novel, Golgi-localized, GTP-binding proteins regulate the transport of APP at the TGN; and (ii) clarify how two genetic risk factors for late-onset AD, both of which are membrane proteins, affect the trafficking of APP. If successful, the proposed studies are expected to increase our understanding of APP sorting and Aß generation, and by extension, elucidate biological processes underpinning AD. Importantly, Dr. Sirkis' F32 training will prepare him for an independent research career that focuses on the intracellular trafficking of proteins involved in neurodegeneration.
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0.976 |