Titel: Imaging of TFG, a protein implicated in neurodegenerative disease, across a variety of scales
Legend: Staining of TFG (green) and a marker of the endoplasmic reticulum (red) in human epithelial cells (left). The localization of TFG was further defined by immunogold electron microscopy (15 nm gold particles), highlighting its accumulation among ER-derived transport carriers (top, right). Single particle electron microscopy was used to determine the structure of recombinant TFG (bottom, right).
Beetz C, Johnson A, Schuh AL, Thakur S, Varga RE, Fothergill T, Hertel N, Bomba-Warczak E, Thiele H, Nürnberg G, Altmüller J, Saxena R, Chapman ER, Dent EW, Nürnberg P, Audhya A. (2013) Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure. Proceedings of the National Academy of Sciences of the United States of America. 110(13):5091-6. .
Johnson A, Bhattacharya N, Hanna M, Pennington JG, Schuh AL, Wang L, Otegui MS, Stagg SM, Audhya A. (2015) TFG clusters COPII-coated transport carriers and promotes early secretory pathway organization. EMBO Journal. 34(6):811-27.
1. Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.
2. In mammalian cells, cargo-laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER-Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII-coated transport carriers traverse a submicron, TFG (Trk-fused gene)-enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three-dimensional electron microscopy reveals the formation of flexible, octameric cup-like structures, which are able to self-associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII-coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII-coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII-coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.
About the lab:
Anjon Audhya’s lab is committed to understanding fundamental mechanisms by which membrane proteins, lipids, and other macromolecules are transported throughout eukaryotic cells. Audhya utilizes interdisciplinary approaches, including biochemistry, structural biology, biophysics, genetics, molecular biology and high resolution fluorescence and electron microscopy.
Additionally, Audhya uses a variety of experimental systems, ranging from simple animal models (e.g. Caenorhabditis elegans) to human induced pluripotent stem cells (iPSCs). He aims to recapitulate individual steps of membrane transport in vitro, using recombinant proteins and chemically defined lipids. The ultimate goal of this research is to identify the regulatory pathways that control membrane deformation, which enable vesicle formation in the endosomal and secretory systems. Although basic research is the cornerstone of his program, Audhya seeks to define pathomechanisms that underlie human disease, focusing on the impact of mutations in key trafficking components that lead to cancer, neurodegeneration, asthma, and diabetes.