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Journal ArticleDOI

Protein mobility in the cytoplasm of Escherichia coli

01 Jan 1999-Journal of Bacteriology (American Society for Microbiology)-Vol. 181, Iss: 1, pp 197-203
TL;DR: Noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli have implications for the understanding of intracellular biochemical networks.
Abstract: The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809‐812, 1997). The apparent diffusion coefficient, Da, of GFP in E. coli DH5a was found to be 7.7 6 2.5 mm 2 /s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with Da of 2.5 6 0.6 mm 2 /s. In addition, GFP mobility can depend strongly on at least two factors: first, Da is reduced to 3.6 6 0.7 mm 2 /s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces Da to 4.0 6 2.0 mm 2 /s. Thus, a single effective cytoplasmic viscosity cannot explain all values of Da reported here. These measurements have implications for the understanding of intracellular biochemical networks.

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Citations
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Journal ArticleDOI
TL;DR: Positive results of crowding include enhancing the collapse of polypeptide chains into functional proteins, the assembly of oligomeric structures and the efficiency of action of some molecular chaperones and metabolic pathways.

2,104 citations

Journal ArticleDOI
TL;DR: This work has shown that a constraint-based reconstruction and analysis approach provides a biochemically and genetically consistent framework for the generation of hypotheses and the testing of functions of microbial cells.
Abstract: Microbial cells operate under governing constraints that limit their range of possible functions. With the availability of annotated genome sequences, it has become possible to reconstruct genome-scale biochemical reaction networks for microorganisms. The imposition of governing constraints on a reconstructed biochemical network leads to the definition of achievable cellular functions. In recent years, a substantial and growing toolbox of computational analysis methods has been developed to study the characteristics and capabilities of microorganisms using a constraint-based reconstruction and analysis (COBRA) approach. This approach provides a biochemically and genetically consistent framework for the generation of hypotheses and the testing of functions of microbial cells.

1,102 citations


Cites background from "Protein mobility in the cytoplasm o..."

  • ...The diffusion rates of macromolecules inside a cell are generally slow and limitin...

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Journal ArticleDOI
TL;DR: A large body of recent experimental evidence for anomalous transport in crowded biological media is reported on in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where a variety of model systems mimic physiological crowding conditions.
Abstract: A ubiquitous observation in cell biology is that the diffusive motion of macromolecules and organelles is anomalous, and a description simply based on the conventional diffusion equation with diffusion constants measured in dilute solution fails. This is commonly attributed to macromolecular crowding in the interior of cells and in cellular membranes, summarizing their densely packed and heterogeneous structures. The most familiar phenomenon is a sublinear, power-law increase of the mean-square displacement (MSD) as a function of the lag time, but there are other manifestations like strongly reduced and time-dependent diffusion coefficients, persistent correlations in time, non-Gaussian distributions of spatial displacements, heterogeneous diffusion and a fraction of immobile particles. After a general introduction to the statistical description of slow, anomalous transport, we summarize some widely used theoretical models: Gaussian models like fractional Brownian motion and Langevin equations for visco-elastic media, the continuous-time random walk model, and the Lorentz model describing obstructed transport in a heterogeneous environment. Particular emphasis is put on the spatio-temporal properties of the transport in terms of two-point correlation functions, dynamic scaling behaviour, and how the models are distinguished by their propagators even if the MSDs are identical. Then, we review the theory underlying commonly applied experimental techniques in the presence of anomalous transport like single-particle tracking, fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). We report on the large body of recent experimental evidence for anomalous transport in crowded biological media: in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where a variety of model systems mimic physiological crowding conditions. Finally, computer simulations are discussed which play an important role in testing the theoretical models and corroborating the experimental findings. The review is completed by a synthesis of the theoretical and experimental progress identifying open questions for future investigation.

1,080 citations


Cites background from "Protein mobility in the cytoplasm o..."

  • ...ense they complement each other. Combining FRAP and a photoactivation technique allowed for a non-invasive measurement of the motion of endogenously expressed GFP in the cytoplasm of E. coli bacteria [250]. The apparent diffusion constant was obtained as 7:7 ±2:5µm2/s at the time scale of some 0.1s, which is about 11 times smaller than for free diffusion in water and signifi- cantly lower than in eukary...

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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.

1,002 citations

Journal ArticleDOI
TL;DR: This work tracks the motion of individual fluorescently labeled mRNA molecules inside live E. coli cells and finds that the motion is subdiffusive, with an exponent that is robust to physiological changes, including the disruption of cytoskeletal elements.
Abstract: We track the motion of individual fluorescently labeled mRNA molecules inside live E. coli cells. We find that the motion is subdiffusive, with an exponent that is robust to physiological changes, including the disruption of cytoskeletal elements. By modifying the parameters of the RNA molecule and the bacterial cell, we are able to examine the possible mechanisms that can lead to this unique type of motion, especially the effect of macromolecular crowding. We also examine the implications of anomalous diffusion on the kinetics of bacterial gene regulation, in particular, how transcription factors find their DNA targets.

764 citations

References
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PatentDOI
17 Aug 1998-Gene
TL;DR: In this article, three classes of GFP mutants having single excitation maxima around 488 nm are shown to be brighter than wild-type GFP following 488-nm excitation.

3,093 citations

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.

2,594 citations


"Protein mobility in the cytoplasm o..." refers methods in this paper

  • ...One of the most useful techniques for studying cytoplasmic diffusion in eukaryotic cells and cell membranes has been the method of “fluorescence recovery after photobleaching” (FRAP) (3, 16)....

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  • ...These bacteria are smaller than the eukaryotic cells previously studied by FRAP, and it is difficult to introduce fluorescently labeled molecules into them....

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  • ...Use of the FRAP technique with image data in the small quasicylindrical geometry of the bacterial cell called for a method of analysis which considers the concentration of GFP throughout the cell rather than only in the photobleached region (see Materials and Methods)....

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  • ...Like fluorochromes used in previous FRAP experiments, GFP can be irreversibly photobleached with sufficiently intense illumination (25)....

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  • ...FRAP experiments have difficulty distinguishing between normal diffusion with a fixed “immobile fraction” and subdiffusion (11)....

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Journal ArticleDOI
TL;DR: Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription.

2,442 citations


Additional excerpts

  • ...In addition, the following E. coli strains transformed with pMGS053 were examined: AB1157 (9), M15(pREP4) (Qiagen), MC1000 (5), MC1061 (5), MG1655 (13), and RP437 (18)....

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  • ...were examined: AB1157 (9), M15(pREP4) (Qiagen), MC1000 (5), MC1061 (5),...

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