Enhanced propagation of motile bacteria on surfaces due to forward scattering

How motile bacteria move near a surface is a problem of fundamental biophysical interest and is key to the emergence of several phenomena of biological, ecological and medical relevance, including biofilm formation. Solid boundaries can strongly influence a cell's propulsion mechanism, thus leading many flagellated bacteria to describe long circular trajectories stably entrapped by the surface. Experimental studies on near-surface bacterial motility have, however, neglected the fact that real environments have typical microstructures varying on the scale of the cells' motion. Here, we show that micro-obstacles influence the propagation of peritrichously flagellated bacteria on a flat surface in a non-monotonic way. Instead of hindering it, an optimal, relatively low obstacle density can significantly enhance cells' propagation on surfaces due to individual forward-scattering events. This finding provides insight on the emerging dynamics of chiral active matter in complex environments and inspires possible routes to control microbial ecology in natural habitats

Mesure des forces d'adhérence cellulaire par microscopie holographique

Mechanical forces, generated by the cell plays crucial role in cell adhesion - common process for different cell lines. ln order to measure the force map during cellular adhesion, we use Traction Force Microscopy (TFM), where cell adheres to the soft substrate in 20 plane, and the forces are calculated from measured displacement field inside the substrate underneath the cell. We built the microscope, where instead of using fluorescent markers, we use spherical polystyrene beads in order to measure the displacement field. Positions of the markers are obtained by analyzing the interference pattern caused by the beads in bright-field light. With this technique, we reach nanometer accuracy of the microsphere position determination, that, respectively, influence accuracy of the calculated force field. With the microscope first measurements were performed with cancer cell line SW 480.Les forces mécaniques, générées par la cellule jouent un rôle crucial dans l'adhésion cellulaire, qui est un processus commun à un grand nombre de lignées cellulaires. Afin de mesurer la champ des forces pendant l'adhérence cellulaire, nous utilisons la microscopie de force de traction, où la cellule adhère à la surface plane d'un substrat souple dans le plan. Les forces sont calculées à partir du champ de déplacement mesuré à l'intérieur du substrat sous la cellule. Nous avons construit le microscope, dans lequel nous utilisons des billes sphériques en polystyrène pour mesurer le champ de déplacement. Les positions des marqueurs sont obtenues en analysant I' image interférentielle des particules. Avec cette technique, nous atteignons une précision nanométrique sur le champ de déplacement des particules, ce qui nous permet d'améliorer la résolution en force de ce type de microscope. Les premières mesures ont été effectuées avec la lignée de cellules cancéreuses SW 480

ColocAnalyzer

ColocAnalyzer - is a program that works with microscopy images. It allows (i) filter images by number of methods, (ii) compute different colocalization coefficients and (iii) apply random spot shuffling method to check whether computed colocalization is random or no

Holographic Traction Force Microscopy

International audienceTraction Force Microscopy (TFM) computes the forces exerted at the surface of an elastic material by measuring induced deformations in volume. It is used to determine the pattern of the adhesion forces exerted by cells or by cellular assemblies grown onto a soft deformable substrate. Typically, colloidal particles are dispersed in the substrate and their displacement is monitored by fluorescent microscopy. As with any other fluorescent techniques, the accuracy in measuring a particule's position is ultimately limited by the number of evaluated fluorescent photons. Here, we present a TFM technique based on the detection of probe particle displacements by holographic tracking microscopy. We show that nanometer scale resolutions of the particle displacements can be obtained and determine the maximum volume fraction of markers in the substrate. We demonstrate the feasibility of the technique experimentally and measure the three-dimensional force fields exerted by colorectal cancer cells cultivated onto a polyacrylamide gel substrate

DNA Nanostructures for Targeted Antimicrobial Delivery.

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle

Diversity of dynamic voltage patterns in neuronal dendrites revealed by nanopipette electrophysiology.

Acknowledgements: The authors thank Elena Dossi, Charles Felix-Calvo, Julien Moulard and Giampaolo Milior for discussion and technical help. J. Mc H. acknowledges funding from AFOSR (Grant No. FA9550-17-1-0118), ANR programme Investissements d'avenir (ANR-10-LABX-54 MEMOLIFE and ANR-IO-IDEX0001-02 PSL Research University) and from FRM (SPF202005011994). D. M. acknowledges funding from Q-life (ANR-17-CONV-0005). G. S. K. S. acknowledges funding from the Michael J Fox Foundation (16238). U. F. K. is supported by ERC Consolidator Grant (DesignerPores n° 647144). D. H. acknowledges funding from the European Research Council (ERC Advanced Grant OrganellenanoComp n° 882673) under the European Union's Horizon 2020 research and innovation programme, Plan Cancer-INSERM (19CS145-00) and ANR (ANR-18-NEUC-0001). N. R. and C. F. K. acknowledge funding from Cam-PSL-French Embassy. N. R. and D. H. acknowledge funding from ANR (ANR-22-CE16-0027) and ANR programme Investissements d'avenir (ANR-10-LABX-54 MEMOLIFE and ANR-IO-IDEX0001-02 PSL Research University). N. R. acknowledges the Service Enseignement Supérieur, Recherche et Innovation de l'Ambassade de France and Churchill College for the French government oversea fellowship.Dendrites and dendritic spines are the essential cellular compartments in neuronal communication, conveying information through transient voltage signals. Our understanding of these compartmentalized voltage dynamics in fine, distal neuronal dendrites remains poor due to the difficulties inherent to accessing and stably recording from such small, nanoscale cellular compartments for a sustained time. To overcome these challenges, we use nanopipettes that permit long and stable recordings directly from fine neuronal dendrites. We reveal a diversity of voltage dynamics present locally in dendrites, such as spontaneous voltage transients, bursting events and oscillating periods of silence and firing activity, all of which we characterized using segmentation analysis. Remarkably, we find that neuronal dendrites can display spontaneous hyperpolarisation events, and sustain transient hyperpolarised states. The voltage patterns were activity-dependent, with a stronger dependency on synaptic activity than on action potentials. Long-time recordings of fine dendritic protrusions show complex voltage dynamics that may represent a previously unexplored contribution to dendritic computations

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.An Open Targets grant [OTAR2054], the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council (MRC), Alzheimer’s Society and Alzheimer’s Research UK

SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.AstraZeneca; Infinitus (China) Ltd

Mutation of the ALS/FTD-associated RNA-binding protein FUS affects axonal development

Aberrant condensation and localisation of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organisation and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signalling. To assess whether loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development