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 diffusion of immature tyrosinase in the endoplasmic reticulum of non-pigmented cells is analyzed by taking advantage of the thermal sensitivity of the tyros in order to suggest that the ER represses random fluctuations of immature TYOSinase molecules while preventing their immobilization.
21 citations
TL;DR: This work presents an agent-based dynamic model using three-dimensional morphologies from electron microscopy tomography which considers the molecular dynamics of the main ATP production components and suggests that internal mitochondrial morphology is not only optimized for ATP production but also provides a mechanism for energy buffering and may represent a mechanisms for cellular robustness.
Abstract: Mitochondria as the main energy suppliers of eukaryotic cells are highly dynamic organelles that fuse, divide and are transported along the cytoskeleton to ensure cellular energy homeostasis. While these processes are well established, substantial evidence indicates that the internal structure is also highly variable in dependence on metabolic conditions. However, a quantitative mechanistic understanding of how mitochondrial morphology affects energetic states is still elusive. To address this question, we here present an agent-based dynamic model using three-dimensional morphologies from electron microscopy tomography which considers the molecular dynamics of the main ATP production components. We apply our modeling approach to mitochondria at the synapse which is the largest energy consumer within the brain. Interestingly, comparing the spatiotemporal simulations with a corresponding space-independent approach, we find minor space dependence when the system relaxes toward equilibrium but a qualitative difference in fluctuating environments. These results suggest that internal mitochondrial morphology is not only optimized for ATP production but also provides a mechanism for energy buffering and may represent a mechanism for cellular robustness.
20 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...…coefficients were estimated previously based on measurements of green fluorescent protein (GFP) in the matrix of mitochondria of diverse cells (Partikian et al., 1998; Dieteren et al., 2011) reporting that the free diffusion is two to fourfold reduced compared to water (Partikian et al.,…...
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...…previously based on measurements of green fluorescent protein (GFP) in the matrix of mitochondria of diverse cells (Partikian et al., 1998; Dieteren et al., 2011) reporting that the free diffusion is two to fourfold reduced compared to water (Partikian et al., 1998; Dieteren et al., 2011)....
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...Effects on diffusion due to the internal structure were studied based on simplified geometries (Partikian et al., 1998; Dieteren et al., 2011) and indicated anomalous diffusion in some cases (Ölveczky and Verkman, 1998), but disagreed on the impact of the internal structure (Dieteren et al., 2011;…...
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...…to the internal structure were studied based on simplified geometries (Partikian et al., 1998; Dieteren et al., 2011) and indicated anomalous diffusion in some cases (Ölveczky and Verkman, 1998), but disagreed on the impact of the internal structure (Dieteren et al., 2011; Partikian et al., 1998)....
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...Although some studies (Scalettar et al., 1991; López-Beltrán et al., 1996; Dieteren et al., 2011) showed evidence of severe hindrance of diffusion in the matrix, more recent experiments estimated that diffusion is only three to four fold smaller than in water (Partikian et al., 1998)....
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TL;DR: It is shown that both full-length proteins are actively transported into the nucleus, and that the homeodomains contain the signals required for this localization and interaction with nuclear components.
Abstract: Double homeodomain (DUX) proteins are encoded by a family of 3.3-kilobase repeated elements dispersed in the human genome. One of these elements named D4Z4 is found in a tandem repeat array on chromosome 4 that is partially deleted in facioscapulohumeral muscular dystrophy. We have evaluated the trafficking and mobility of two DUX proteins, DUX1 and DUX4. We transfected C2C12 myoblasts with cDNA encoding these proteins fused to the green fluorescent protein and studied their intracellular localization and diffusional mobilities using fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We also studied truncated forms of the proteins, containing one or both homeodomains or a region outside the homeodomains. We show that both full-length proteins are actively transported into the nucleus, and that the homeodomains contain the signals required for this localization. DUX1 is more mobile than DUX4 within the nucleus (t1/2 = 4.8 s for DUX1 and 13.4 s for DUX4), suggesting differen...
19 citations
TL;DR: The major phenomenological manifestations of mitochondrial age-related dysfunction including biochemical, regulatory and energetic features are reviewed and it seems unlikely that a single linear cause and effect relationship between any specific aspect of mitochondrial biology and ageing can be established.
Abstract: Mitochondrial dysfunction is associated with ageing, but the detailed causal relationship between the two is still unclear. We review the major phenomenological manifestations of mitochondrial age-related dysfunction including biochemical, regulatory and energetic features. We conclude that the complexity of these processes and their inter-relationships are still not fully understood and at this point it seems unlikely that a single linear cause and effect relationship between any specific aspect of mitochondrial biology and ageing can be established in either direction.
19 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...An obvious effect of the dynamic remodeling of the network is the redistribution of mitochondrial components, including lipids [394], proteins [395] and mitochondrial DNA [396]....
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TL;DR: It is concluded that juxtaposition of the mitochondria and the Ca2+ release sites is crucial for rapid signal transmission to maintain cardiac-energy balance and the idealized 3D model of cardiac excitation-contraction and metabolism is a powerful tool to study cardiac energetics.
19 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.
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