Macromolecular crowding: obvious but underappreciated.

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.

Genome-scale models of microbial cells: evaluating the consequences of constraints

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.

https://iopscience.iop.org/article/10.1088/0034-4885/76/4/046602/pdf

Anomalous transport in the crowded world of biological cells.

Macromolecular crowding: an important but neglected aspect of the intracellular environment.

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.

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Physical nature of bacterial cytoplasm.

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.

/pdf/protein-mobility-in-the-cytoplasm-of-escherichia-coli-3xv5565l88.pdf

Pierre Etienne Wolf

Papers

Protein mobility in the cytoplasm of Escherichia coli