The long-term goals of the currently funded project are to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER), known as ER exit sites. At these locations, COPII-coated carriers are first generated, packaging cargoes for transport to ER- Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to ER exit sites. The COPII coat is composed of two multimeric protein complexes, Sec23/24 and Sec13/31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII vesicle biogenesis and anterograde transport in cells. Our currently funded project focuses on the role of TFG, a metazoan-specific protein required for the normal trafficking of COPII-coated transport carriers. Based on our preliminary results, we hypothesize that TFG forms a highly regulated meshwork at the ER/ERGIC interface that facilitates the local retention of ER-derived vesicles, providing sufficient time for COPII coat disassembly and subsequent fusion with ERGIC membranes. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, including hereditary spastic paraplegia (HSP), suggesting a role for COPII-mediated transport in maintaining neuron function. This revision application seeks to expand the scope of our project. Specifically, our goal is to engineer new rat models of HSP and define mechanisms by which mutations in key organelle components, including TFG (SPG57), atlastin-1 (SPG3A) and the ESCRT-I subunit Vps37A (SPG53), lead to axonopathy in the corticospinal tract. These studies leverage our previous successes in using CRISPR-mediated genome editing in rats and seeks to define both shared and unique pathological features observed as a result of mutations that impact ER and endosome homeostasis, which are known to underlie neurodegenerative disease in patients.
The directed movement of proteins and membranes between different subcellular locations is a fundamental process required for the proper functioning of all eukaryotic cells. Many neurodegenerative diseases including hereditary spastic paraplegias can be caused by axonal transport defects. The proposed research will determine how organelle trafficking and homeostasis are appropriately regulated, enhancing our fundamental understanding of these processes, which should facilitate the future identification of therapeutic targets for disease intervention.
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