Year: 2016
Type: dissertation
Abstract: Inter-organelle communication is a key feature of basic eukaryotic cells functions and of numerous cell signaling events (Bravo-Sagua et al., 2014). One of the best-characterized interorganelle cross talk is that between Endoplasmic Reticulum (ER) and mitochondria(Lopez-Crisosto et al., 2015). Also referred to as mitochondria associated ER-membranes (MAMs), the existence of mitochondria-ER contacts was uncovered 50 years ago through electron microscopic studies(Copeland and Dalton, 1959). The 90s saw the first break-through in functional significance of the MAMs, when Vance and Rizzuto et al., showed that exchange of phospholipids and calcium transfer occurs between the two organelles(Rizzuto et al., 1998; Vance, 1990). Despite the importance of the tether in metabolism and disease(Cali et al., 2013; Giorgi et al., 2015; Vance, 2014), only few proteins have so far been described to maintain the structural integrity of the tether in mammals (Lopez-Crisosto et al., 2015). MFN2was the firststructural tether to be identified. ER localized MFN2 forms homo and heterotypic interactions with mitochondrialMFN2 and MFN1, thus tethering the two organelles. As residual juxtapositionexist in Mfn2-/-cells, yet to be discovered tethersmust exist (de Brito and Scorrano, 2008). Hence, we set out to perform a genome wide screening to identify the structural components of the tether in mouse embryonic fibroblasts (MEFs). In order to perform the genome wide screening, we capitalized on the FRET based biosensor developed by Csordas et al., where CFP fused with FRB domain and YFP fused with FKBP domain were targeted to ER (by a Sac 1 signaling sequence) and mitochondria (by an Akap signaling sequence) respectively (Csordas et al., 2010). We modified this biosensor by driving expression of both fluorescent molecules from a single mRNA, introducing between their cDNAs a self-cleaving Tav2A peptide(Luke et al., 2008). This modification allows expressing equimolar level of the proteins. Thanks to FKBP and FRB binding domain, which interacts upon addition of rapamycin, we were able to study the basal and maximal level of juxtaposition between the two organelles. We then utilized this modified biosensor to perform a high content screen to identify molecules that either tether or space ER and mitochondria. We termed them as tethers and spacers respectively. We analyzed raw images from the screen and calculated the maximum number of contacts possible for every gene. Following automated image analysisand statistical analysis using R programming in cellHTS2 package of the primary screen performed on ~10,000 genes, we identified 14.4% genes as ER-mitochondria tethers (i.e., genes that once ablated increase the distance between the two organelles) and 5.3% genes as spacers (i.e., genes that once ablated decrease the distance between the two organelles). Pathway analysis using reactome and Panther predicted both existing and new pathways that are yet to explored in terms of ERmitochondria communication. Subcellular localization analysis of the tethers revealed eight proteins predicted to be present in both ER and outer mitochondrial membrane (OMM). One of the eight is myotonic dystrophy protein kinase (DMPK) and we have further characterized how DMPK tethers ER and mitochondria. DMPK is mutated in myotonic dystrophy 1 (DM1) (Cho and Tapscott, 2007). Having six known isoforms based on alternate splicing in humans and mouse (Wansink et al., 2003), DMPK’s role in myotonic dystrophy is not yet understood completely since the KO model of this gene developed late onset of myopathy and cardiac contractile dysfunction (Reddy et al., 1996). The subcellular localization either to ER or to mitochondria of the isoforms have been identified, although very less emphasis has been given to the functional relevance of different sub-cellular localizations. In conclusion, we have developed a new method to assess the proximity of ER and mitochondria and we have utilized this technology in a high content screen to identify novel structural components of the tether. The data from the screen has also spanned several existing pathways where ER-mitochondria junction has been implicated but not completely understood. Hence, the molecules identified from our screen, will help in unraveling the molecular players in these cellular pathways.
Author: Sowmya Lakshminarayanan
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: European Commission, Generalitat de Catalunya, Università degli Studi di Padova, Institució Catalana de Recerca i Estudis Avançats, Associazione Italiana per la Ricerca sul Cancro, Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas, National Institutes of Health, Instituto de Salud Carlos III, National Heart, Lung, and Blood Institute, FP7 Ideas: European Research Council
Awards: G1551068933, G3261704812, Molecular Anatomy and Pathophysiology of the endoplasmic reticulum-mitochondria interface, G452027594, MITOCHONDRIAL FUSION FACTORS AND CARDIOMYOPATHY, Extending the Optic atrophy 1 dependent cristae remodeling: from models to a rationale for therapy of autosomal dominant optic atrophy
OpenAlex ID: W2554364930