TY - JOUR
T1 - A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators
AU - Schauss, Astrid C.
AU - Huang, Huiyan
AU - Choi, Seok Yong
AU - Xu, Liqun
AU - Soubeyrand, Sébastien
AU - Bilodeau, Patricia
AU - Zunino, Rodolfo
AU - Rippstein, Peter
AU - Frohman, Michael A.
AU - McBride, Heidi M.
N1 - Funding Information:
This work was supported by CIHR MOP#43935 to H.M.M. and a CIHR Postdoctoral fellowship to ACS. The authors thank members of the laboratory and Luca Pellegrini (University of Laval) for a critical reading of the manuscript.
PY - 2010/7/26
Y1 - 2010/7/26
N2 - Background: Mitochondria 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.Results: In 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.Conclusions: The 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.
AB - Background: Mitochondria 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.Results: In 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.Conclusions: The 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.
UR - http://www.scopus.com/inward/record.url?scp=77955099425&partnerID=8YFLogxK
U2 - 10.1186/1741-7007-8-100
DO - 10.1186/1741-7007-8-100
M3 - Article
C2 - 20659315
AN - SCOPUS:77955099425
SN - 1741-7007
VL - 8
JO - BMC Biology
JF - BMC Biology
M1 - 100
ER -