Single molecule/particle tracking analysis program SMTracker 2.0 reveals different dynamics of proteins within the RNA degradosome complex inBacillus subtilis

Abstract

Single-molecule (particle) tracking is a powerful method to study dynamic processes in cells at highest possible spatial and temporal resolution. We have developed SMTracker, a graphical user interface for automatic quantifying, visualizing and managing of data. Version 2.0 determines distributions of positional displacements in x- and y- direction using multi-state diffusion models, discriminates between Brownian, sub- or superdiffusive behaviour, and locates slow or fast diffusing populations in a standardized cell. Using SMTracker, we show that the Bacillus subtilis RNA degradosome consists of a highly dynamic complex of RNase Y and binding partners. We found similar changes in molecule dynamics for RNase Y, CshA, PNPase and enolase, but not for phosphofructokinase, RNase J1 and J2, to inhibition of transcription. However, the absence of PfkA or of RNase J2 affected molecule dynamics of RNase Y-mVenus, indicating that these two proteins are indeed part of the degradosome. Molecule counting suggests that RNase Y is present as a dimer in cells, at an average copy number of about 500, of which 46% are present in a slow-diffusive state and thus likely engaged within degradosomes. Thus, RNase Y, CshA, PNPase and enolase likely play central roles, and RNase J1, J2 and PfkA more peripheral roles, in degradosome architecture.

Y-Complex Proteins Show RNA-Dependent Binding Events at the Cell Membrane and Distinct Single-Molecule Dynamics

“…Thus, the components of the Y-complex behave similarly towards rifampicin treatment, corresponding to the finding of complex formation by the three proteins [18]. It has been shown that RNase Y still forms foci at the cell membrane after inhibition of transcription [20], but that RNase Y relocalizes from the membrane towards to cytosol [32]. Because there is a dispute over whether SMT data on RNase Y are valid, we will perform additional control experiments before further proceeding with a comparison between RNase Y and Y-complex protein dynamics in a future study.…”

“…For Northern blot analysis, RNA samples were mixed with 2× PAGE loading dye (1× TBE in formamide containing xylene cyanol and bromphenol blue). 32 P-labelled HR RiboRuler Ladder (Thermo Fisher Scientific, Walthman, MA, USA) was prepared using 20 µl HR RiboRuler Ladder, 40 µCi of γ-32 P-ATP (Hartmann Analytik GmbH, Braunschweig, Germany), 1× Buffer A (Thermo Fisher Scientific, Walthman, MA, USA) and 20 U PNK (Thermo Fisher Scientific, Walthman, MA, USA) at 37 • C for 1 h. Total RNA was fractionated by agarose gel electrophoresis (1.2% agarose, 0.6% formaldehyde v/v, 1× MOPS buffer, pH 7) at 110 V. Using a capillary transfer, RNA was transferred from the agarose gel to Whatman Nytran SuPerCharge nylon blotting membrane (GE Healthcare, Chicago, IL, USA) in 10× SSC buffer, pH 7 overnight. The membrane was UV cross-linked and prehybridized in 20 ml ROTI ® Hybri-Quick (Carl Roth, Karlsruhe, Germany) for 30 min at 45 • C. 10 µl of previously radiolabeled RNA riboprobes were added to the prehybridized membrane and incubated for 3 h at 45 • C. The blots were washed twice for 20 min with wash solution 1 (2× SSC, 0.1% SDS w/v) and twice for 20 min with wash solution 2 (0.1× SSC, 0.1% SDS w/v) at 45 • C. Results were visualized using storage phosphor screens (GE Healthcare, Chicago, IL, USA) and Typhoon TM biomolecular imager (GE Healthcare, Chicago, IL, USA).…”

Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein

“…To identify differences in protein mobility and/or behavior, the resulting tracks were subjected to dwell time, mean-squared-displacement (MSD), and square displacement (SQD) analysis in SMTracker 2.0 [ 49 ], described previously [ 50 ].…”

“…Exponentially growing cells of B. subtilis (left/cyan panels) (BHF073) or C. glutamicum (right/magenta panels) (B6G8) expressing a DivIVA-Halo fusion, respectively, were stained with TMR (HaloTag ligand). Individual protein trajectories were recorded via single-molecule localization microscopy and analyzed using Trackmate [ 48 ] and the SMTracker [ 49 ] software packages. ( A ) Representative cells of B. subtilis and C. glutamicum displaying confined (blue) and fast (red) trajectories of DivIVA-Halo.…”

“…Exponentially growing cells of different B. subtilis strains (BHF073 and BHF074) expressing the indicated DivIVA-Halo fusion, respectively, were stained with TMR (HaloTag ligand). Individual protein trajectories were recorded via single-molecule localization microscopy and analyzed using Trackmate [ 48 ] and the SMTracker [ 49 ] software packages. ( A ) Cartoon of DivIVA and MinJ in a dividing cell.…”