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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01 Nov 2004
2 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...…which are responsible for the formation of COPI vesicles (Presley et al., 2002; Elsner et al., 2003), the mobility of proteins in the mitochondria (Partikian et al., 1998), the lumen of the endoplasmic reticulum (ER) (Dayel et al., 1999), on ER and Golgi membranes (Cole et al., 1996), in the…...
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..., 2003), the mobility of proteins in the mitochondria (Partikian et al., 1998), the lumen of the endoplasmic reticulum (ER) (Dayel et al....
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01 Jan 2014
TL;DR: Key players in many metabolic and signaling pathways are nucleotides such as ATP and GTP that have a crucial role in cytoskeleton-mediated events and the sensing power of dynamic macromolecular associations is discussed.
Abstract: Cells face the enormous challenge of generating a single phenotype that must be coherent with myriad internal and external conditions. For such phenotypes to have multifarious but meaningful outputs entails the sensing, and integration of a wide variety of chemical and physical information, hence the coordination of metabolic and signaling processes. This sensing, integration, and coordination are carried out by the complex ultrastructural arrays and moonlighting functions of the cytoskeletal network. In the cellular context, the direction and potency of sensing are determined by the structure-related responses of the cytoskeletal network to the activity of individual macromolecules in conjunction with associated metabolites and nucleotides. These responses comprise the binding (hetero-association) of these macromolecules to the cytoskeleton and the consequences of this binding on the behavior of both partners, among them the stability and dynamics of the cytoskeleton, and the catalytic and regulatory properties of the individual proteins (and/or their specific complexes). The latter is of specific importance in regulation at a high level of organization via the formation of microcompartments in linear pathways or at metabolic crossroads. In addition, key players in many metabolic and signaling pathways are nucleotides such as ATP and GTP that have a crucial role in cytoskeleton-mediated events. These issues are illustrated with examples, and the sensing power of dynamic macromolecular associations is discussed.
2 citations
Dissertation•
13 Dec 2016
TL;DR: CRISPR-mediated endogenous tagging was crucial to investigate the localization, dynamics and abundance of mitochondrial prohibitin 1 and 2, PHB1 and PHB2, in human cells and uncovered the hitherto unknown organization of individual prohibitins into clusters.
Abstract: Ectopic overexpression of fluorescent fusion proteins for live cell imaging studies often leads to a multitude of artefacts, but protein expression at endogenous levels in mammalian cells was difficult to achieve so far. To avoid common problems associated with overexpression, this study used the CRISPR-Cas9 genome engineering system for site-specific endogenous protein tagging in human cells. First, a general workflow for genome editing was established and then applied to generate heterozygous and homozygous human knock-in cells that express a fluorescent fusion from a genomic locus. Three human genes (HMGA1, VIM and ZYX) were tagged with the reversibly switchable fluorescent protein rsEGFP2 and the benefit of endogenous over ectopic expression demonstrated using flow cytometry and confocal microscopy. Moreover, low light intensity RESOLFT super-resolution microscopy could be applied to study nanoscale protein dynamics at physiologically relevant protein expression levels in living knock-in cells.
CRISPR-mediated endogenous tagging was crucial to investigate the localization, dynamics and abundance of mitochondrial prohibitin 1 and 2, PHB1 and PHB2, in human cells. While overexpression of PHB1 and PHB2 caused aberrant mitochondria, endogenous tagging of prohibitins with the fluorescent protein Dreiklang (DK) restored wildtype mitochondrial morphology. Overexpression of PHB2-DK and human estrogen receptor α caused a mislocalization of PHB2-DK in the nucleus of HeLa cells, but also this artefact was not observed in endogenously tagged PHB2-DK HeLa cells. Homologous recombination frequencies for PHB1 and PHB2 tagging were remarkably high and a number of heterozygous PHB1-DK and PHB2-DK knock-in clones could be generated. STED super-resolution microscopy uncovered the hitherto unknown organization of individual prohibitins into clusters. Dual-color STED imaging demonstrated a colocalization of tagged and untagged PHB1 and PHB2 indicating that PHB complex formation is not affected by protein tagging. Intriguingly, the vast majority of prohibitins is found at the mitochondrial cristae membrane where they form exceptionally static protein assemblies. Surprisingly, the global amount of PHB2 was found to be 4-5 times higher than that of PHB1, which is in contrast to in vitro studies conducted on purified yeast prohibitins. After integrating the results on prohibitin localization and abundance with morphological data about the ultrastructural organization of mitochondria and under the assumption that human prohibitins form a 1 MDa complex, it was estimated that about 31-36 individual PHB complexes occupy a single cristae membrane.
2 citations
Cites result from "Rapid Diffusion of Green Fluorescen..."
...This observation is in accordance with previous reports that showed a high mobility of matrix localized GFP (Partikian et al., 1998)....
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Dissertation•
01 Jan 2008
2 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
..., 2001), the diffusion coefficient and residence time of proteins in the Golgi, organelle lumen, cytosol, and plasma membrane (Cole et al., 1996; Dayel et al., 1999; Hirschberg et al., 1998; Nehls et al., 2000; Partikian et al., 1998)....
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...…2004; Presley et al., 2002; Raiborg et al., 2006; Wu et al., 2001), the diffusion coefficient and residence time of proteins in the Golgi, organelle lumen, cyto- sol, and plasma membrane (Cole et al., 1996; Dayel et al., 1999; Hirschberg et al., 1998; Nehls et al., 2000; Partikian et al., 1998)....
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TL;DR: An algorithm for digital processing of images recorded by means of scanning laser fluorescent tomography is developed in this article, where images of the test object fabricated from a polymethylmethacrylate matrix with rhodamin 6G put in water solution of dry milk modeling a brain tissue are processed.
Abstract: An algorithm for digital processing of images recorded by means of scanning laser fluorescent tomography is developed. Images of the test object fabricated from a polymethylmethacrylate matrix with rhodamin 6G put in water solution of dry milk modeling a brain tissue are processed. The algorithm allows not only the contours of the object distorted because of strong scattering in biological tissues but also a finer object structure to be reconstructed. In addition, the algorithm enables three-dimensional object structure to be reconstructed for the known distribution of dye concentration in the object, which is also demonstrated in the present paper.
2 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.
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