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.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

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.

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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.