Biology subjects

Bureau, L.

Publications and source records attributed to Bureau, L..

4 recordsLinked to original sources

bioRxiv2024

Optical sectioning for reflection interference microscopy

Reflection Interference Contrast Microscopy (RICM, also known as interference reflection microscopy) and related techniques have become of wide interest to the biophysical, soft matter and biochemistry communities owing to their exquisite sensitivity for characterising thin films or individual nanoscopic objects adsorbed onto surfaces, or for monitoring cell-substrate interactions. Over the recent years, striking progresses have been made to improve the sensitivity and the quantitative analysis of RICM. Its use in more complex environments, with spurious reflections stemming from a variety of structures in the sample, remains however challenging. In this paper, we demonstrate two optical sectioning methods that effectively reduce such background and can be readily implemented in a conventional RICM setup: line confocal detection, and structured illumination microscopy. We characterise experimentally the benefits to image quality and demonstrate the use of the methods for quantitative imaging of complex biological and biomimetic samples: cellular membranes, thin organic films, surface biofunctionalization. We then discuss the benefits of each method and provide guidelines to arbitrate between sectioning and signal-to-noise ratio. Finally, we provide a detailed description of our experimental setup and a home-written image acquisition and processing software that should allow the interested reader to duplicate such a setup on a home-built or commercial microscope.

biophysics↗

bioRxiv2024

Uncovering the simple adhesive strategy of the Toxoplasma parasite for high-speed motility

Toxoplasma gondii is a protozoan parasite that has evolved a developmental morphotype called tachyzoite that navigates between cells and moves in and out of them in a wide repertoire of homeothermic hosts. Relying on a uniquely constant apicobasal bipolarity coupled to an actomyosin-driven retrograde surface flow, the tachyzoite has elaborated a molecular machinery to assemble transient anchoring contacts with the environment, which support the traction force required to power a typical helical gliding motility. Combining micropatterning with live, reflection interference contrast and expansion microscopies, we bring first nanoscale evidence that the tachyzoite needs to build only one apical anchoring contact with the substrate, thus spatially defining a minimal force transmission platform over which it can slide. We uncover that the apicobasal driven surface flow is set up in response to extracellular biochemical cues independent of adhesin release and tachyzoite-surface interactions, hence prior to motile activity. Furthermore, to identify the minimal adhesion requirements for helical gliding at the level of individual molecular species, we combine biochemical and biophysical quantitative assays based on tunable surface chemistry and quartz crystal microbalance with dissipation monitoring. These approaches uncover that glycosaminoglycan (GAG)-parasite interactions are sufficient to promote a productive contact for helical gliding and pave the way for the characterization of the structure and density of the molecules functionally engaged at this essential parasite-substrate mechanosensitive interface.

cell biology↗

bioRxiv2023

Competitive specific anchorage of molecules onto surfaces: quantitative control of grafting densities and contamination by free anchors

The formation of surfaces decorated with biomacromolecules such as proteins, glycans or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes, and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest, and present effective guidelines towards optimizing incubation conditions for the quantitative control of surface densities. Focussing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

biophysics↗

bioRxiv2022

Substrate stiffness impacts early biofilm formation via a modulation of twitching motility

Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments, and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials. Here, we demonstrate experimentally that the promiscuous opportunistic pathogen Pseudomonas aeruginosa explores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation.

biophysics↗