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Journal ArticleDOI

Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

23 Feb 1998-Journal of Cell Biology (The Rockefeller University Press)-Vol. 140, Iss: 4, pp 821-829

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.

AbstractIt 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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Citations
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Journal ArticleDOI
TL;DR: Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components.
Abstract: Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.

1,174 citations


Journal ArticleDOI
04 Apr 2003-Science
TL;DR: The development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells are traced.
Abstract: The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.

985 citations


Journal ArticleDOI
TL;DR: It is proposed that the addition of crowding agents should become as routine as controlling pH and ionic strength if the authors are to meet the objective of studying biological molecules under more physiologically relevant conditions.
Abstract: Biological macromolecules have evolved over billions of years to function inside cells, so it is not surprising that researchers studying the properties of such molecules, either in extracts or in purified form, take care to control factors that reflect the intracellular environment, such as pH, ionic strength and composition, redox potential and the concentrations of relevant metabolites and effector molecules. There is one universal aspect of the cellular interior, however, that is largely neglected--the fact that it is highly crowded with macromolecules. It is proposed that the addition of crowding agents should become as routine as controlling pH and ionic strength if we are to meet the objective of studying biological molecules under more physiologically relevant conditions.

921 citations


Journal ArticleDOI
TL;DR: The results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding.
Abstract: The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (Dw) decreased from 53 × 10−8 to 0.81 × 10−8 cm2/s for sizes of 21–6000 bp; D w was related empirically to DNA size:D w = 4.9 × 10−6cm2/s·[bp size]−0.72. DNA diffusion coefficients in cytoplasm (D cyto) were lower than D w and depended strongly on DNA size.D cyto/D w decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was 2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.

731 citations


Journal ArticleDOI
TL;DR: The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH and suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonations and conformational changes at lower pH.
Abstract: It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pK a values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and ∼1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5–8, the fluorescence spectral shape, lifetime (2.8ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in 5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH 4 Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.

669 citations


Cites background from "Rapid Diffusion of Green Fluorescen..."

  • ..., 1997) and mitochondria (Partikian et al., 1998) by fluorescence photobleaching and time-resolved fluorescence methods....

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References
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Journal ArticleDOI
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.
Abstract: Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments Information obtainable from FPR experiments includes: (a) identification of transport process type, ie the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, ie the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G

2,479 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…...

    [...]


Journal ArticleDOI
06 Sep 1996-Science
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.

2,133 citations


Journal ArticleDOI
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.

1,453 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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Journal ArticleDOI
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.
Abstract: In 1970 Riggs et al. (1) reported that Escherichia coli lac repressor binding to λ DNA in vitro seemed to find its target (operator) site on the DNA at a rate as much as 1000-fold faster than the upper limit estimated for a diffusion-controlled process involving macromolecules of this size. This observation startled and intrigued many physically oriented molecular biologists and biochemists and initiated a flurry of theoretical and experimental papers seeking to offer an explanation. However, scrutiny of the older literature reveals that scientists, ranging from mathematicians to biologists, had long been concerned with how systems of various sorts might transcend the rate limits set by three-dimensional diffusion control (2). Such problems are now of interest at many different levels. The pure physical chemist feels that an understanding of such phenomena might provide new insight into what happens when molecules meet and rearrange in the course of forming and passing through the transition state complex. The enzyme mechanician hopes that the secrets of some of the astonishing increases in rates achieved in enzyme-catalyzed reactions may be revealed by a study of these rate accelerations. And the cell biologist who studies macromolecular interactions and assembly processes is intrigued by the possibility that these systems may reveal opportunities for acceleration of intracellular rates beyond the limits set by the relatively slow diffusion of macromolecules in the cytoplasm. In this minireview we propose to touch on recent progress in all of these areas but will focus primarily on a problem that has engaged our attention over the past few years, i.e. how do protein regulators of gene expression at the transcriptional level find their regulatory DNA targets at speeds that appear to be faster than diffusion controlled?

962 citations