New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction.

Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins.

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

Gelsolin-like activation of villin: calcium sensitivity of the long helix in domain 6.

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1-V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin's functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-Kd calcium binding in V6 (Kd1 of 22 µM and Kd2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism.