Heterogeneity of Subcellular Diffusion in Bacteria Based on Spatial Segregation of Ribosomes and Nucleoids.

It has long become clear that in spite of generally lacking internal membrane systems, bacteria contain well-structured subcellular structures of usually filamentous proteins, and a preferred 3D arrangement of their chromosome(s). Some of these systems are set up by so-called cytoskeletal elements, or by polar landmark proteins, but the mechanism of specific localization is still unclear in most cases. Intriguingly, apart from such spatially organizing systems, the bacterial cytoplasm has unusual properties in terms of the diffusion of molecules, which varies between different sites within the cell. In many bacteria, chromosomes are compacted into centrally located nucleoids, being orderly folded as opposed to consisting of random coils of DNA. In these bacteria, there is a separation of transcription and translation, such that transcription by RNA polymerase occurs on the nucleoids, and translation takes place mostly at the cell poles and directly underneath the cell membrane, because 70S ribosomes accumulate at sites surrounding the nucleoids. Interestingly, accumulation of ribosomes appears to slow down diffusion of enzymes, noticeable for larger enzyme complexes, while nucleoids provide areas of confined motion for DNA-binding proteins, yet acceleration zones for non-DNA-binding proteins. Crowded regions at the cell poles set up zones of higher concentration of the translation machinery, shortening diffusion distances for rate-limiting translation factor/ribosome interactions, and of metabolic enzymes, possibly speeding up pathways containing low concentrations of metabolites. Thus, heterogeneous diffusion adds another layer of subcellular organization on top of cytoskeletal elements.

The Acetyltransferase RibT From Bacillus subtilis Affects in vivo Dynamics of the Multimeric Heavy Riboflavin Synthase Complex.

Flavins are ubiquitous molecules in life as they serve as important enzyme cofactors. In the Gram-positive, soil-dwelling bacterium Bacillus subtilis, four well-characterized gene products (the enzymes RibDG, RibE, RibAB, and RibH) catalyze the biosynthesis of riboflavin (RF) from guanosine-triphosphate (GTP) and ribulose-5-phosphate (R5P). The corresponding genes form an operon together with the gene ribT (ribDG-E-AB-H-T), wherein the function of this terminal gene remained enigmatic. RibT has been structurally characterized as a GCN5-like acetyltransferase (GNAT), however, with unidentified target molecules. Bacterial two-hybrid system revealed interactions between RibT, RibH, and RibE, forming the heavy RF synthase complex. Applying single particle tracking (SPT), we found that confined (sub)diffusion of RibT is largely dependent on interacting RibE and, to a lesser degree, on interacting RibH. By induced expression of otherwise low-expressed ribT from an ectopic locus, we observed a decrease in the subpopulation considered to represent capsids of the heavy RF synthase and an increase in the subpopulation thought to represent pentamers of RibH, pointing to a putative role for RibT in capsid disassembly. Complementarily, either deletion of ribT or mutation of a key residue from RibH (K29) suspected to be the substrate of RibT for acetylation leads to increased levels of subpopulations considered as capsids of RibH-mVenus (RibH-mV) in comparison to wild-type (wt)-like cells. Thus, we provide evidence for an indirect involvement of RibT in RF biosynthesis by a putative capsid disassembling mechanism considered to involve acetylation of RibH residue K29 at the three-fold symmetry axis of 60-mer capsids.

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.

Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm.

BACKGROUND: Knowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo. RESULTS: We have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles. CONCLUSIONS: Our results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

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

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.

Photon correlation versus interference of single-atom fluorescence in a half-cavity.

Photon correlations are investigated for a single laser-excited ion trapped in front of a mirror. Varying the relative distance between the ion and the mirror, photon correlation statistics can be tuned smoothly from an antibunching minimum to a bunchinglike maximum. Our analysis concerns the non-Markovian regime of the ion-mirror interaction and reveals the field establishment in a half-cavity interferometer.

Feedback cooling of a single trapped ion.

Based on a real-time measurement of the motion of a single ion in a Paul trap, we demonstrate its electromechanical cooling below the Doppler limit by homodyne feedback control (cold damping). The feedback cooling results are well described by a model based on a quantum mechanical master equation.