Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy |
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Authors: | Werner JH Koopman Mark A Hink Sjoerd Verkaart Henk-Jan Visch Jan AM Smeitink |
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Institution: | a Department of Membrane Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands b Microscopical Imaging Centre, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands c Department of Paediatrics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands d MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands |
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Abstract: | We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYFP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When CI activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility. |
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Keywords: | ACF autocorrelation function CI complex I or NADH:ubiquinone oxidoreductase F degree of mitochondrial length and of branching FCS fluorescence correlation spectroscopy IMM inner mitochondrial membrane mitoEYFP enhanced yellow fluorescent protein targeted to the mitochondrial matrix Nc number of mitochondria per cell ROI region of interest Δψ mitochondrial membrane potential |
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