Abstract
BackgroundMitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques.ResultsIn order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction.ConclusionsThe adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99
Highlights
Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function
Generation of a bimolecular complementation marker pair for the quantification of mitochondrial fusion In order to develop a high-throughput mitochondrial fusion assay, we adapted a bimolecular complementation strategy that would allow multiple outputs [15]. cDNAs were generated encoding mitochondrial targeted split Venus and Renilla luciferase separated by a leucine zipper
Fusion between mitochondria expressing each of these chimeric proteins would lead to their dimerization through the leucine zipper motifs, promoting the functional assembly of both luciferase and Venus yellow fluorescent protein (YFP) [16,17]
Summary
A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators. Astrid C Schauss, Huiyan Huang, Seok-Yong Choi, Liqun Xu1, Sébastien Soubeyrand, Patricia Bilodeau, Rodolfo Zunino, Peter Rippstein, Michael A Frohman, Heidi M McBride1*
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