Semsey, S. | bioRxiv Science

bioRxiv2020

Filopodia rotate and coil by actively generating twist in their actin shaft

Filopodia are actin-rich structures, present on the surface of practically every known eukaryotic cell. These structures play a pivotal role in specific cell-cell and cell-matrix interactions by allowing cells to explore their environment, generate mechanical forces, perform chemical signaling, or convey signals via intercellular tunneling nano-bridges. The dynamics of filopodia appear quite complex as they exhibit a rich behavior of buckling, pulling, length and shape changes. Here, we find that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling of their actin rich core. Importantly, we show the rotational dynamics of the filamentous actin inside filopodia for a range of highly distinct and cognate cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist, and hence rotation, is an emergent phenomenon of active filaments confined in a narrow channel which points to a generic mechanism present in all cells. Our measurements confirm that filopodia exert traction forces and form helical buckles in a range of different cell types that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

biophysics↗

bioRxiv2017

Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage {varphi}80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules in E. coli persister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.\n\nImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

microbiology↗