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 2006
TL;DR: Methods for automatic trajectory classification, detection of motion within trajectories, and adaptive data encoding to maximize classification p erformance are developed and applied and used in two studies of virus motion on the plasma membrane of live cells.
Abstract: Diffusion processes constitute a key mechanism for transpo rt in biological cells. Nutrition, organization, growth, and signal transduction in cells are largely determined by diffusion mechanisms. The complex three-dimension al shapes of intracellular structures and the confinement of certain molecules to membranes however complicate the experimental analysis and computational si mulation of diffusion in live cells. This thesis is concerned with the development and implementation of computational methods to analyze, model, and simulate di ffusion processes in realistic cell environments. In cell biology, mobile particles such as molecules, ions, v esicles, or viruses diffuse within the confines of the cell geometries. Two cases ar considered: individually tracked particles, and their mean collective mo tion. The former case entails single particle tracking methods to directly follo w the motion of individual particles. We present an accurate and computationally effic ient image processing algorithm to determine trajectories of moving particles fr om digital videos. These trajectories are then analyzed with respect to their motion pr perties. We extend existing analysis methods to cases of anomalous diffusion a nd show that both the speed and the confinement of the motion can be quantified indep e ntly. Automated trajectory analysis enables high throughput rat es, minimizes human bias, and increases reproducibility. We therefore develop and apply methods for automatic trajectory classification, detection of motion p atterns within trajectories, and adaptive data encoding to maximize classification p erformance. The developed computational tools are used in two studies of virus motion on the plasma membrane of live cells. Analysis and simulation of the collective motion of abundan t particles is based on continuum theory, which yields a model equation for the ev olution of the resulting concentration field. Solving this governing equatio n s challenging for realistically complex cell geometries. We present particle met hods to handle these complex geometries, and extend them to computations of diff usion on curved and moving surfaces. The capability of numerically simulating diffusion both in spaces and on surfaces of complex shape allows to investigate the accuracy of fluorescence recovery iv experiments. We present for the first time the measurement of molecular diffusion constants in the endoplasmic reticulum of live cells by taking into account the complex geometry of the organelle. All methods are implemented on the basis of a newly developed software library for hybrid particle-mesh simulations on parallel computer s. The library is presented in this thesis and its parallel efficiency and scalabilit y are demonstrated on a range of test cases.
17 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...Diffusion constants of GFP and GFP-tagged prote ins have been reported for the cytoplasm [278], the nucleus [216], the ER [75, 203], mitochondria [210], the Golgi complex [60, 254], and for different membranes in t he cell [89, 90, 186]....
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TL;DR: This chapter summarizes recent developments in the field of quantitative simulation approaches and frameworks for the description of intracellular transport processes and special focus is laid on compartmented and spatiotemporally resolved simulation approaches.
Abstract: The complexity and internal organization of mammalian cells as well as the regulation of intracellular transport processes has increasingly moved into the focus of investigation during the past two decades. Advanced staining and microscopy techniques help to shed light onto spatial cellular compartmentation and regulation, increasing the demand for improved modeling techniques. In this chapter, we summarize recent developments in the field of quantitative simulation approaches and frameworks for the description of intracellular transport processes. Special focus is therefore laid on compartmented and spatiotemporally resolved simulation approaches. The processes considered include free and facilitated diffusion of molecules, active transport via the microtubule and actin filament network, vesicle distribution, membrane transport, cell cycle-dependent cell growth and morphology variation, and protein production. Commercially and freely available simulation packages are summarized as well as model data exchange and harmonization issues.
16 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...Diffusion measurements of unconjugated GFP in mitochondria ðDGFP;mito 1⁄4 2 3 10 7 cm2 s 1Þ [106] indicate similar free diffusion characteristics, while the apparent diffusion in the endoplasmic reticulum (ER) is decreased approximately...
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TL;DR: A sixth generation amino terminated polyamide polylysine dendrimer which is fluorescent allowed the study of nuclear uptake and mobility in living lung carcinoma (SK/MES-1) and colon adenocarcinoma (Caco-2) cells.
15 citations
TL;DR: In this paper, the authors investigate diffusive search on planar networks, motivated by tubular organelle networks in cell biology that contain molecules searching for reaction partners and binding sites, and find that global structural properties -the total edge length and number of loops - are sufficient to largely determine network exploration times.
Abstract: We investigate diffusive search on planar networks, motivated by tubular organelle networks in cell biology that contain molecules searching for reaction partners and binding sites. Exact calculation of the diffusive mean first-passage time on a spatial network is used to characterize the typical search time as a function of network connectivity. We find that global structural properties - the total edge length and number of loops - are sufficient to largely determine network exploration times for a variety of both synthetic planar networks and organelle morphologies extracted from living cells. For synthetic networks on a lattice, we predict the search time dependence on these global structural parameters by connecting with percolation theory, providing a bridge from irregular real-world networks to a simpler physical model. The dependence of search time on global network structural properties suggests that network architecture can be designed for efficient search without controlling the precise arrangement of connections. Specifically, increasing the number of loops substantially decreases search times, pointing to a potential physical mechanism for regulating reaction rates within organelle network structures.
15 citations
TL;DR: It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.
Abstract: Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.
14 citations
Cites background or methods from "Rapid Diffusion of Green Fluorescen..."
...Analogously to what done in the cytoplasm, this hypothesis was tested by FRAP and anisotropy-decay measurements using GFP as inert tracer targeted to the mitochondrial matrix of fibroblasts, liver, skeletal muscle and epithelial cell lines [82]....
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...Overall, data suggest that metabolite channeling may not be required to overcome diffusive barriers, while clustering of matrix enzymes in membrane-associated complexes may serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse [82]....
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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