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 Jan 2016
TL;DR: In this paper, the effect of serum from calorie-restricted rats versus serum from rats fed ad libitum, diluted tenfold in the medium, which did not contribute significantly to the pool of nutrients, on β-cell mitochondrial function and dynamics under regular and high-nutrient culture conditions was studied.
Abstract: β‐cells quickly adjust insulin secretion to oscillations in nutrients carried by the blood, acting as fuel sensors. However, most studies of β‐cell responses to nutrients do not discriminate between fuel levels and signaling components present in the circulation. Here we studied the effect of serum from calorie‐restricted rats versus serum from rats fed ad libitum, diluted tenfold in the medium, which did not contribute significantly to the pool of nutrients, on β‐cell mitochondrial function and dynamics under regular and high‐nutrient culture conditions. Insulin secreting beta‐cell derived line (INS1) cells incubated with serum from calorie‐restricted rats (CR serum) showed higher levels of peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC‐1α) and active nitric oxide synthase. The expression of mitofusin‐2 (Mfn‐2) and optic atrophy 1 (OPA‐1), proteins involved in mitochondrial fusion, was increased, while the levels of the mitochondrial fission mediator dynamin related protein 1 (DRP‐1) were reduced. Consistent with changes in mitochondrial dynamics protein levels, CR serum treatment increased mitochondrial fusion rates, as well as their length and connectivity. These changes in mitochondrial morphology were associated with prolonged glucose‐stimulated insulin secretion and mitochondrial respiration. When combining CR serum and high levels of glucose and palmitate (20 and 0.4 mm, respectively), an in vitro model of type II diabetes, we observed that signaling promoted by CR serum was enough to overcome glucolipotoxicity, as indicated by CR‐mediated prevention of mitochondrial fusion arrest and reduced respiratory function in INS1 cells under glucolipotoxicity. Overall, our results provide evidence that non‐nutrient factors in serum have a major impact on β‐cell mitochondrial adaptations to changes in metabolism.
1 citations
01 Jan 2012
TL;DR: This study investigated the mechanisms of clustering of the apoptosis receptor CD95 after induction with its ligand and the oligomerization level before induction and proposed a model where the receptor is present as monomers or dimers at physiological concentrations in the absence of induction.
Abstract: In this study I investigated the mechanisms of clustering of the apoptosis receptor CD95
after induction with its ligand and the oligomerization level before induction. Previous
studies demonstrated that receptor clustering is an essential part in apoptosis signaling.
Mostly using biochemical methods and nuclear magnetic resonance the essential role of
various mechanisms in receptor clustering could be shown, such as lipid raft localization of
the receptor, clustering via the receptor death domain and internalization. An oligomerization
of receptors before induction via a so called pre-ligand assembly domain could be
shown at high concentrations, so that typically the receptor is assumed to be trimeric
before induction. Moreover a consistent model of the role of all mechanisms in different
cell types and direct observation on the plasma membrane is still missing. Therefore
I investigated receptor clustering in single living cells on the cell membrane using microscopy.
In particular I quantified intensities to correlate with the number of molecules.
and studied receptor clustering by dynamics measurements using fluorescence recovery
after photobleaching. I observed that receptor clustering is concentration dependent and
in HeLa at high receptor concentrations the ligand as only crosslinking mechanism yields
mean clusters size of seven molecules. Death domain assembly and lipid raft localization do not effect clustering in this cell type. At physiological expression levels the pre-ligand
assembly shows an oligomer distribution with an eminent part of monomers or dimers.
By knockdown of the receptor the distribution can be pushed to monomers. I made a
consistent study for the type II cell line HeLa, considering many proposed clustering
mechanisms and showed that ligand binding alone is a strong mechanism that can lead
to high levels of clustering. I propose a model where the receptor is present as monomers
or dimers at physiological concentrations in the absence of induction.
1 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...diffusion coefficients in cellular compartments or on membranes: free movement, hindered diffusion [84] [87] [94] [90] [89] [88]...
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...could show with FRAP experiments, that this hindrance ”relatively mild” and proteins can diffuse nearly as freely in the matrix as in aqueous solution [87]....
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...in the mitochondrial matrix [87], in the nucleus [88], in the cytoplasm, in the ER-lumen [89] andin the ER- and Golgi membranes [90] While previous considerations and studies ”suggest that the mitochondrial matrix, if it is indeed a crowded protein solution, would severely hinder solute diffusion” Partikian et....
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TL;DR: In this paper, the authors verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy.
Abstract: Mitochondria are highly dynamic organelles that interchange their contents mediated by fission and fusion. However, it has previously been shown that the mitochondria of cultured human epithelial cells exhibit a gradient in the relative abundance of several proteins, with the perinuclear mitochondria generally exhibiting a higher protein abundance than the peripheral mitochondria. The molecular mechanisms that are required for the establishment and the maintenance of such inner-cellular mitochondrial protein abundance gradients are unknown. We verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy. We found that the Tom20 gradients are established immediately after cell division and require the presence of microtubules. Furthermore, the gradients are abrogated in hyperfused mitochondrial networks. Our results suggest that inner-cellular protein abundance gradients from the perinuclear to the peripheral mitochondria are established by the trafficking of individual mitochondria to their respective cellular destination.
1 citations
Dissertation•
23 Feb 2011
TL;DR: Evidence is provided that after clustering of the receptors, the mobility of diacylated probes such as those found in Src-family of kinases, was reduced and this immobilisation was found to be insensitive to cholesterol depletion, arguing against a role for conventional 'lipid rafts' in the initiation of receptor signalling.
Abstract: Elements that are foreign to the human body, such as bacteria, viruses and fungi, are recognised by cells of the innate immune system. Through a process termed phagocytosis, microorganisms are bound, internalised and destroyed. In this thesis, we focus upon how host cells respond to IgG-opsonised targets, studying both the initial stages of Fc-receptor (FcR) ligation and the later stages of phagocytic cup formation. We provide evidence that after clustering of the receptors, the mobility of diacylated probes such as those found in Src-family of kinases, was reduced. This immobilisation was found to be insensitive to cholesterol depletion, arguing against a role for conventional ‘lipid rafts’ in the initiation of receptor signalling. Furthermore, decreased mobility was only partially dependent upon the presence of actin which could provide a physical restriction. Importantly, inhibiting Src-family kinase activity, completely abrogated immobilisation. These results are highly suggestive of a previously unrecognised mechanism for the initation of FcR signalling.
1 citations
TL;DR: In this paper , the inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration, and it has been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology.
Abstract: The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.
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