Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria

Abstract

Willems PH. Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria. Am J Physiol Cell Physiol 294: C1124-C1132, 2008. First published March 19, 2008 doi:10.1152/ajpcell.00079.2008.-Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient cells than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity.

Mammalian Mitochondrial Complex I: Biogenesis, Regulation, and Reactive Oxygen Species Generation

“…This reactivity, as reflected by its affinity toward electrons (E h ¼ 0.94 V), is higher than that of H 2 O 2 (E h ¼ 0.32 V), but much lower than that of OH · (E h ¼ 2.31 V) (101). Additionally, the mitochondrial matrix has a higher viscosity than water, thereby slowing diffusion (144,147). Therefore, in a cellular environment, it is to be expected that the Dx for O ·À 2 is (much) smaller than 0.16 mm.…”

“…To allow quantitative assessment of the effect of rotenone treatment on mitochondrial motility and intramatrix protein diffusion, we developed a novel approach to follow the mobility of a matrix-targeted variant of GFP (mitoEYFP) in living cells by means of fluorescence correlation spectroscopy (FCS) (144,147). FCS is a technique that measures intensity fluctuations caused by single fluorescent molecules moving through a small detection volume.…”

“…Crucially, successful FCS analysis requires that mitoEYFP expression levels are not too high. To achieve the latter, mitoEYFP was introduced into the cells by using a modified (insect) baculoviral transfection approach (144,147). FCS analysis revealed that the fraction of moving mitochondria and their speed were reduced in cells cultured in presence of 100 nM rotenone for 72 h. The intramatrix diffusion of mitoEYFP, however, was increased in both stationary and moving mitochondria in rotenone-treated cells (144).…”

Solute diffusion is hindered in the mitochondrial matrix

“…Next, FP mobility is measured using FCS (fluorescence correlation spectroscopy) or FRAP (fluorescence recovery after photobleaching). This is then followed by curve fitting and/ or mathematical modeling of the experimental data to obtain the diffusion constant of the FP (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, these analysis methods generally do not include realistic (i.e., experimentally determined) information concerning the spatial dimensions and nanostructure of the compartment.…”

“…Moreover, the temporal scale of most FRAP models does not quantitatively match with that of FRAP experiments. Therefore it was already recognized some time ago (8)(9)(10)(11)(12)(13)(14)(15)(16)(17) that the above approaches will only yield an "apparent" (biased) value for the diffusion constant (D app ) of a given FP, which represents an underestimation of the "real" (i.e., purely solvent-dependent) diffusion constant (D solvent ).…”