105 research outputs found
Salt-dependent rheology and surface tension of protein condensates using optical traps
An increasing number of proteins with intrinsically disordered domains have
been shown to phase separate in buffer to form liquid-like phases. These
protein condensates serve as simple models for the investigation of the more
complex membrane-less organelles in cells. To understand the function of such
proteins in cells, the material properties of the condensates they form are
important. However, these material properties are not well understood. Here, we
develop a novel method based on optical traps to study the frequency-dependent
rheology and the surface tension of PGL-3 condensates as a function of salt
concentration. We find that PGL-3 droplets are predominantly viscous but also
exhibit elastic properties. As the salt concentration is reduced, their elastic
modulus, viscosity and surface tension increase. Our findings show that salt
concentration has a strong influence on the rheology and dynamics of protein
condensates suggesting an important role of electrostatic interactions for
their material properties.Comment: 5 pages, 3 figures, 1 supplemen
Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition
Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function
Increased airway glucose increases airway bacterial load in hyperglycaemia.
Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection. We hypothesize that increased airway glucose caused by hyperglycaemia leads to increased bacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisation is significantly more likely when blood glucose is high. We engineered mutants in genes affecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa, strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented with glucose as the sole carbon source. The effect of glucose on growth in vivo was tested using streptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose. Bacterial burden in hyperglycaemic animals was greater than control animals when infected with wild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reduced both airway glucose and bacterial load. These data support airway glucose as a critical determinant of increased bacterial load during diabetes
Identification of Biofilm-Associated Cluster (bac) in Pseudomonas aeruginosa Involved in Biofilm Formation and Virulence
Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a ΔpA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729–pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster
Shildrick’s monster: exploring a new approach to difference/disability through collective biography
Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains
Evolution des populations de Leptosphaeria maculans sous l'effet de gènes de résistance introgresses dans le colza à partir de moutardes
International audienc
- …