Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix
TL;DR: 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.
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TL;DR: The application of FRAP is extended to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle and the parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.
49 citations
Cites methods from "Rapid Diffusion of Green Fluorescen..."
...Earlier, the 1D approach was used for measurement of diffusion in mitochondrial matrix [14] and is common in application to Escherichia coli [78–80]....
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TL;DR: An MSTO1 loss‐of‐function mutation is associated with a human disorder showing mitochondrial involvement and M STO1 likely has a physiologically relevant role in mitochondrial morphogenesis by supporting mitochondrial fusion.
Abstract: The protein MSTO1 has been localized to mitochondria and linked to mitochondrial morphology, but its specific role has remained unclear We identified a c22G > A (pVal8Met) mutation of MSTO1 in patients with minor physical abnormalities, myopathy, ataxia, and neurodevelopmental impairments Lactate stress test and myopathological results suggest mitochondrial dysfunction In patient fibroblasts, MSTO1 mRNA and protein abundance are decreased, mitochondria display fragmentation, aggregation, and decreased network continuity and fusion activity These characteristics can be reversed by genetic rescue Short‐term silencing of MSTO1 in HeLa cells reproduced the impairment of mitochondrial morphology and dynamics observed in the fibroblasts without damaging bioenergetics At variance with a previous report, we find MSTO1 to be localized in the cytoplasmic area with limited colocalization with mitochondria MSTO1 interacts with the fusion machinery as a soluble factor at the cytoplasm‐mitochondrial outer membrane interface After plasma membrane permeabilization, MSTO1 is released from the cells Thus, an MSTO1 loss‐of‐function mutation is associated with a human disorder showing mitochondrial involvement MSTO1 likely has a physiologically relevant role in mitochondrial morphogenesis by supporting mitochondrial fusion
49 citations
Cites methods from "Rapid Diffusion of Green Fluorescen..."
...Using confocal microscopy, time series of fluorescence images were recorded and 25 lm2 square-shaped areas were illuminated by a pulsed laser to photoactivate mtPA-GFP (Eisner et al, 2014; Weaver et al, 2014), which is a soluble protein that shows rapid diffusion (Partikian et al, 1998)....
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TL;DR: The pivotal FRAP studies are reviewed, which gave rise to current models of complex cell dynamics, leading to a fundamental rethinking of long-held theories of cellular dynamism.
46 citations
TL;DR: The need for the development of mitochondria-specific drug and DNA delivery systems is discussed, and the currently employed strategies for mitochondrial drug targeting are evaluated, including some of their potential therapeutic applications.
Abstract: Mitochondrial dysfunction causes or contributes to a large number of human disorders including neuromuscular and neurodegenerative diseases, diabetes, ischaemia-reperfusion injury and cancer. Increasing efforts are being made towards mitochondria-directed pharmacological intervention, leading to the emergence of 'mitochondrial medicine' as a new field of biomedical research. The identification of new molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch new therapies for the treatment of mitochondria-related diseases, based either on the selective protection, repair or eradication of cells. This review discusses the need for the development of mitochondria-specific drug and DNA delivery systems, and evaluates the currently employed strategies for mitochondrial drug targeting, including some of their potential therapeutic applications.
46 citations
Cites background or methods or result from "Rapid Diffusion of Green Fluorescen..."
...Likewise, measurement of the rotation of GFP by time-resolved anisotropy gave a rotational correlation time very similar to that of GFP in water [53]....
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...Such a high density of enzymes and proteins makes the mitochondrial matrix the most crowded aqueous cellular compartment, and it has been suggested by several authors (reviewed in [53]) that the diffusion of small solutes such as metabolites and enzymes, might, therefore, be severely restricted....
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...The quantitative analysis of their bleach data using a mathematical model of matrix diffusion gave diffusion coefficients for GFP only three- to fourfold smaller than that for GFP diffusion in water [53]....
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...It has also been proposed that biochemical metabolites are passed between enzyme and enzyme complexes by a special type of channelling mechanism, which would be independent of any aqueous-phase diffusion process, and that mitochondrial water is in an organised state which might even change the basic physical chemistry of enzyme reactions (reviewed in [53])....
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...Verkman’s group has measured via spot photobleaching the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of four different cell lines [53]....
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TL;DR: Photoconversion of green fluorescent protein (GFP) is established as an optical ‘highlighter’ to investigate the continuity of the mitochondrial matrix in living budding yeast (Saccharomyces cerevisiae) and induces a several 100‐fold increase in red fluorescence of matrix targeted GFP without affecting cell viability.
44 citations
References
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TL;DR: The theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments are presented and some model experiments on aqueous solutions of rhodamine 6G are described.
2,594 citations
"Rapid Diffusion of Green Fluorescen..." refers background in this paper
...As discussed by Axelrod et al. (1976) for conventional two-dimensional spot photobleaching, this approximation is reasonably valid for practical laser/lens systems; the same considerations would apply for bleaching of long thin mitochondria where bleach profile is nearly constant across the thin…...
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TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Abstract: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
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TL;DR: The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 Å by multiwavelength anomalous dispersion phasing methods and the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.
Abstract: The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of s-sheet with an α-helix inside and short helical segments on the ends of the cylinder. This motif, with s-structure on the outside and α-helix on the inside, represents a new protein fold, which we have named the s-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr86 with reduction of its Cα-Cs bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.
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"Rapid Diffusion of Green Fluorescen..." refers background in this paper
...The three–amino acid chromophore in GFP is fixed rigidly within a barrel structure (Yang et al., 1996; Örmo et al., 1996)....
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TL;DR: This minireview has attempted to provide some overall perspective on the question of how various forms of diffusion in reduced dimensions, or diffusion within a nonspecifically bound state, can speed biological interactions beyond the limits normally set by three-dimensional diffusion processes.
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