We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli . We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Introduction: Gene therapy is a breakthrough medical field which focuses on the therapeutic delivery of recombinant nucleic acids in order to treat or prevent a broad spectrum of diseases. However, a number of important obstacles remain before its wide introduction into clinical practice can be envisaged. One of the biggest bottlenecks is the lack of efficient and safe delivery technologies, particularly, for in vivo distribution. Above and beyond standard requirements for carriers, the delivery systems for gene therapy ideally use a hit-and-run principle (to minimize off-target effect and display of immunogenic moieties). None of the currently used viral vectors fulfills all of these requirements. Therefore, the growing variety of non-viral delivery platforms represents a promising alternative.Areas covered: This review summarizes the Layer-by-Layer (LbL) approaches that can be effectively used for the gene delivery, considering various examples with the transfer of pDNA, mRNA, siRNA as well as genome-editing tools. Ex vivo gene modification of clinically relevant cells and clinical aspects for possible application of LbL systems in gene therapy are also underlined. Expert opinion:The LbL technique provides broad opportunities for the delivery of genetic material for various purposes and offers promise for future clinical application in gene therapy.

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We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to infer the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells. We find that this observation is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.