Single-molecule Dynamics and Localization of mRNAs, ribosomal Protein L1 and the membrane remodeling protein DynA in Bacillus subtilis
In this work, single-molecule tracking, in combination with SMTracker software, is the main microscopy method to generate and analyze data. This allows various processes to be studied in a millisecond time range with a high optical resolution in living cells/bacteria. Due to new findings, including...
Fluoreszenzmikroskopie mRNA-Dynamiken mRNA-Lokalisation Ribosom Dynamik DynA Dynamik DynA Lokalisation Membranstress
single-molecule tracking mRNA mRNA dynamics mRNA localization mRNA labeling ribosomal assembly ribosome dynamics DynA dynamics membrane remod
mRNA Einzelmolekülverfolgung Chemie Bacillus ribosomales Protein L1 DynA MS2-System Transkription und Translation Ribosom
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|In this work, single-molecule tracking, in combination with SMTracker software, is the main microscopy method to generate and analyze data. This allows various processes to be studied in a millisecond time range with a high optical resolution in living cells/bacteria. Due to new findings, including improved methods, a number of theories that were previously considered established have to be reconsidered. This also applies to protein biosynthesis in bacteria. A temporal and spatial separation of transcription and translation of these two processes is becoming increasingly likely at least in some bacteria. However, the extent to which this occurs is still unclear. Two general models exist for this, which attempt to explain where mRNA translation takes place and whether this is a process separate from transcription or coupled to it: 1.) The mRNA remains close to its transcription site, with the bacterial chromosome acting as a template - where both, coupling of transcription and translation and separation, are possible - or 2.) mRNAs localize where the protein to be encoded is later required. Transcription and translation are thus rather spatially and temporally separated from each other. Importantly, these models may apply differently to different bacterial species. This work focuses mainly on the question of where translation of different mRNAs takes place in the Gram-positive bacterium Bacillus subtilis and what dynamics they exhibit. For this work, the already often used RNA labelling method, the MS2-system, was used, which had to be modified a little in advance in order to achieve optimal results. The results show that already one and two repeats of the MS2-binding sequence are sufficient to detect mRNAs with the MS2-system. In this context, it could be shown that unspecific binding of the MS2-binding protein occurs and affects the growth of cells expressing it, regardless of the co-expression of the associated binding site. Nevertheless, detection of mRNAs is still possible. To address the question of where translation occurs in B. subtilis, mRNAs encoding soluble, membrane and extracellular proteins were used. The localization of these mRNAs provides insight into where translation might take place. The results of this work do not fit exclusively to either model 1) or model 2), but have features of both models. All tested mRNAs show a localization at the poles and the surrounding of the nucleoid, whereas mRNAs encoding membrane proteins show a tendency towards the membrane. The co-expression and co-localization of a mRNA, ypbR-ypzF, and one of the proteins encoded from it, DynA, supports the assumption that a mRNA and its protein product could colocalize. This investigation further includes the dynamics of mRNAs, which previous studies have mainly determined in an indirect or theoretical way. The understanding of how mRNAs move in the cell is best explained by assuming at least two, possibly three populations, with a static population likely representing the translation of mRNA, a possible transition complex with the ribosomal subunits, as well as a freely diffusing population existing if three populations are assumed. The square displacement analysis also shows that the diffusion constants and thus the speed of the mRNAs appear to be size-independent and that mRNAs can move anywhere in the cell within a few seconds, although they are noticeably slower than cytosolic proteins. The assembly of the ribosome also plays an important role here and is probably already indirectly reflected by the mRNA study. The data generated of the ribosomal protein of the 50S subunit, by using single-molecule tracking further supports this. In addition to its localization, for the first time in a Gram-positive bacterium, its dynamics were investigated. Here, more than three populations can be assumed, whereby the static population, which represents a possible translation, corresponds to the static population of the investigated mRNAs. In addition, a fast, freely diffuse population can be observed, as well as the formation of a transition complex with several intermediates, which argues for the dynamic, complex structure of the ribosome and agrees with the mRNA data. Furthermore, the localization data match and support the results obtained from the mRNAs.
The bacterial dynamin-like protein, DynA from B. subtilis, was not only used for co-localization analyses with its mRNA transcript, but was also further characterized. This protein is involved in cell division, where it plays an important role in membrane fusion of the newly divided cells. In addition, cells lacking DynA are less resistant to phage attack and membrane stress induced by chemical reagents. However, the type of membrane stress responsible for DynA recruitment is not fully understood. Using epifluorescence microscopy and single-molecule tracking, I was able to show that DynA only responds to specific stresses, probably induced pores in the membrane caused by attacking components of lipid II. Furthermore, by studying the dynamics of DynA, three different populations of mobility could be found. A static population that is involved in membrane fusion, including during cell division and repair of membrane damage - a process that occurs relatively quickly -, a slow mobile population that searches for possible membrane damage, and a population for probably freely diffusing DynA molecules that has a cytoplasmic localization not known so far. Importantly, even a slight change in the number of DynA molecules is enough to repair the damage caused by the antibiotics.