Super-resolving microscopy reveals the localizations and movement dynamics of stressosome proteins in Listeria monocytogenes

Abstract

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.

Dynamic structure of E. coli cytoplasm: supramolecular complexes and cell aging impact spatial distribution and mobility of proteins

“…Detailed protocol can be found in 1 , 47 . Briefly, diluted to OD 600 0.05 overnight MBM preculture (see Culturing conditions paragraph) was grown for 5 h at 30 °C with shaking at 200 rpm until culture density of ~0.15.…”

“…Selected 250×250 pixels fields of view were illuminated with temporally controlled 405 and 561 nm laser pulses as described in 1 and 47 . In short, 405 nm laser pulse (OBIS 405 LX, 50 mW max.…”

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

“…Importantly, the translational diffusion of the selected proteins is location-dependent in E. coli , with the cell poles displaying slower diffusion throughout the whole set of investigated proteins and one pole showing faster diffusion than the other. , The extent of the slowdown in the pole regions exceeds the confining effects of the cell membrane boundary, as inferred from computer simulations, and instead is most likely a consequence of hindrance by large macromolecular complexes due to accumulation of damaged proteins primarily at the old cell pole (Figure ). Preliminary experiments on protein diffusion in the Gram-positive pathogen L. monocytogenes point toward a similar location-dependent mobility. , It still is an outstanding question whether symmetrically dividing unicellular microorganisms age . The differences in diffusion coefficients and protein probe concentrations between old and new cell poles suggest that exclusion of aggregates and other supramolecular complexes from the nucleoid leads to bacterial aging .…”

“…143 Preliminary experiments on protein diffusion in the Gram-positive pathogen L. monocytogenes point toward a similar locationdependent mobility. 105,174 It still is an outstanding question whether symmetrically dividing unicellular microorganisms age. 175 The differences in diffusion coefficients and protein probe concentrations between old and new cell poles suggest that exclusion of aggregates and other supramolecular complexes from the nucleoid leads to bacterial aging.…”

Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli

“…Despite the advancements made in the field of single molecule fluorescence microscopy 16,17,[35][36][37][38][39] , diffusion measurements are s.ll highly influenced by the effect of confinement, especially in small compartments such as the bacterial cytoplasm 16 and periplasm 40,41 , and eukaryo,c organelles 42 . Here, diffusion coefficients near the cell boundaries always appear lower than in the cell center 16,38 .…”