Abstract
Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state of the cell. In this protocol, we describe a method to analyze mitochondrial structure in body wall muscles in the nematode Caenorhabditis elegans, using both transgenic and dye-based approaches.
Keywords: C. elegans, Mitochondria, Calcium, Mitochondrial membrane potential, TMRE, Mitotracker
Background
itochondria are involved in ATP production, cellular respiration, calcium buffering and reactive oxidative species (ROS) metabolism (Brookes et al., 2004). Mitochondrial structure and function are dynamic and closely linked, therefore analyzing mitochondrial structure can provide clues to the status of mitochondrial health (Sarasija and Norman, 2015). We developed two sets of protocols to assess mitochondrial structure in the body wall muscle of Caenorhabditis elegans. In the first protocol, we used transgenic ccIs4251 strain in which GFP is targeted to the matrix of the body wall muscle mitochondria to visualize the mitochondria (Fire et al., 1998). In the second protocol, we used mitochondrially targeted dyes, MitoTrackerTM Red CMXRos and tetramethylrhodamine ethyl ester (TMRE) to determine the structural integrity of the body wall muscle mitochondria. Normally, animals used in in-vivo imaging are anesthetized, however anesthetizing the animals could lead to mitochondrial morphological changes (Han et al., 2012), complicating data analysis. Our protocols allow for the in-vivo imaging of mitochondrial structure in live, un-anaesthetized nematodes.
Materials and Reagents
Equipment
Software
Part I: Determining mitochondrial structure using transgenic lines
Procedure
Data analysis
Part II: Determining mitochondrial structural and functional integrity using mitochondrial targeted fluorescent dyes
Notes
Recipes
Note: For additional information, please refer to He, 2011.
Acknowledgments
The authors would like to acknowledge J. T. Laboy for her help in ordering and preparing reagents, and P. McKeown-Longo, Y. Tang, M. Barroso and their lab members for support. Some nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). The Alzheimer’s Association (NIRG-09-132122) and NIH (GM088213) supported this work. The authors declare that there are no conflicts of interest or competing interests.
References
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