Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix
TLDR
The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers, and it is proposed that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.Abstract:
It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.read more
Citations
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Journal ArticleDOI
Dynamics of mitochondrial RNA-binding protein complex in Trypanosoma brucei and its petite mutant under optimized immobilization conditions.
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TL;DR: This work represents the first attempt to examine how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism in live trypanosomes in the single, large mitochondrion of Trypanosoma brucei.
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Regulation of signal transduction by spatial parameters: a case in NF– κ B oscillation
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Mitochondrial Quality Control in Hepatocellular Carcinoma.
TL;DR: In this paper, the influence of mitochondria on the biological behavior of hepatocellular carcinoma (HCC) was reviewed and the role and mechanism of interaction between different levels of mitochondrial quality control in regulating HCC occurrence and progression as well as resistance development.
Dissertation
Dynamique de l'agrégation protéique chez la bactérie Escherichia coli
TL;DR: In this article, a double approche theorique et experimentale, basee sur la modelisation and la microscopie time-lapse and microfluidique, is presented.
Book ChapterDOI
Diffusion Measurements by Fluorescence Recovery After Photobleaching
TL;DR: Fluorescence recovery after photobleaching (FRAP) is a classic technique for measurement of the translational diffusion of fluorophores and fluorescently labeled macromolecules and is used widely for determination of solute/macromolecule diffusion in membranes and aqueous compartments, as well as for semiquantitative analysis of protein transport mechanisms.
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