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: Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by Procollagen C‐proteinase and proteolytic modification of its allosteric regulator, PCOLCE1 led to a 20–30‐ and 3–6‐fold increased collagen deposition in two‐ and three‐dimensional cultures, respectively.
146 citations
TL;DR: The theory and practice of performing photobleaching studies using a confocal microscope are examined and measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions are illustrated.
Abstract: The wide availability of the confocal microscope and the emergence of green fluorescent protein (GFP) transfection technology has led to the increasing use of photobleaching studies to examine aspects of cellular dynamics in living cells. In this review, we examine the theory and practice of performing photobleaching studies using a confocal microscope. We illustrate the application of photobleaching protocols using our own measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions and examine other examples from the literature.
141 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...A number of studies have demonstrated the feasibility of performing FRAP measurements to investigate diffusion of soluble components in the lumen of the mitochondria and ER (Olveczky and Verkman 1998; Partikian et al. 1998; Dayel et al. 1999)....
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...Partikian et al. (1998) found that the rate of diffusion of a GFP fusion protein in the mitochondrial lumen was similar to that in the cell cytoplasm, i.e. 3- to 4-fold lower than in water....
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TL;DR: A review of the recent results showing the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations, and the kinetic and structural properties of these metabolic pathways in order to channel NADH to the inner mitochondrial membrane are summarized.
Abstract: Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpd1p, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.
133 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...However, it has been shown that matrix-addressed GFP is able to diffuse rapidly, suggesting that other matrix proteins are organized in membrane-bound complexes, which lets a relatively uncrowded aqueous space allowing the rapid diffusion of metabolites [50]....
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TL;DR: It is proposed that the accuracy in evaluating FRAP measurements can be improved by means of accompanying computer simulations, as current efforts to approach biological problems with biophysical models have to rely on experimentally determined model parameters.
Abstract: Confocal fluorescence recovery after photobleaching (FRAP) is today the prevalent tool when studying the diffusional and kinetic properties of proteins in living cells. Obtaining quantitative data for diffusion coefficients via FRAP, however, is challenged by the fact that both bleaching and scanning take a finite time. Starting from an experimental case, it is shown by means of computer simulations that this intrinsic temporal limitation can lead to a gross underestimation of diffusion coefficients. Determining the binding kinetics of proteins to membranes with FRAP is further shown to be severely hampered by additional diffusional contributions, e.g. diffusion-limited binding. In some cases, the binding kinetics may even be masked entirely by diffusion. As current efforts to approach biological problems with biophysical models have to rely on experimentally determined model parameters, e.g. binding rates and diffusion constants, it is proposed that the accuracy in evaluating FRAP measurements can be improved by means of accompanying computer simulations.
132 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...the shuttling of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus (15–17), the kinetics of peripheral membrane proteins which are responsible for the formation of COPI vesicles (18,19), as well as the diffusional motion of proteins in the cytoplasm (20), the mitochondria (11), the lumen of the ER (9), and on ER and Golgi membranes (21)....
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TL;DR: Mitochondrial fusion is frequent in skeletal muscle, and its disruption jeopardizes excitation–contraction coupling and may contribute to the pathology of myopathies.
Abstract: Genetic targeting experiments indicate a fundamental role for mitochondrial fusion proteins in mammalian physiology. However, owing to the multiple functions of fusion proteins, their related phenotypes are not necessarily caused by altered mitochondrial fusion. Perhaps the biggest mystery is presented by skeletal muscle, where mostly globular-shaped mitochondria are densely packed into the narrow intermyofilamental space, limiting the interorganellar interactions. We show here that mitochondria form local networks and regularly undergo fusion events to share matrix content in skeletal muscle fibers. However, fusion events are less frequent and more stable in the fibers than in nondifferentiated myoblasts. Complementation among muscle mitochondria was suppressed by both in vivo genetic perturbations and chronic alcohol consumption that cause myopathy. An Mfn1-dependent pathway is revealed whereby fusion inhibition weakens the metabolic reserve of mitochondria to cause dysregulation of calcium oscillations during prolonged stimulation. Thus, fusion dynamically connects skeletal muscle mitochondria and its prolonged loss jeopardizes bioenergetics and excitation–contraction coupling, providing a potential pathomechanism contributing to myopathies.
132 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...Mitochondrial matrix is predominantly composed of soluble proteins that show rapid diffusion (Partikian et al., 1998)....
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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.
2,232 citations
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.
1,502 citations
"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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1,070 citations
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.
1,017 citations