SoFTmicroDRoP

The dynamics and ageing of complex protein condensates are extensively investigated, in a biomedical context (prion-like pathological assemblies in RNA-protein granules) or for applications (high-dose formulations of therapeutic antibodies and stabilizing agents). Condensates formed by association of flexible chains, sp. polyelectrolytes, with proteins exhibit features that lie between gel-like networks and colloidal dispersions. Experimental studies face challenges due to limited amount of (recombinant) proteins and difficulties in preparing homogeneous solid-like samples, without the help of thermal annealing that may degrade the proteins. Condensates can be formed as microdroplets of liquid coacervates, whose transition toward solidification requires gradual changes of density and composition. To this aim, we propose to manipulate microvolumes (< 1 microliter, initially fluid condensates) either in custom microfluidic chips enabling dialysis, or by slow uniform drying in an ad hoc rheometer cell (slow diffusion of water vapor). Microdialysis facilitates homogeneization via a sequence of coalescence and rigidification transition controled by successive fast equilibrations with external solutions (pH, salt variations) and enables forward/backward variations to assess reversibility. We will explore representative fibrillation and aggregation-prone models (a-syn, mAb) whose phase separation depends on tunable associations with polyanionic or polycationic peptides (polyLys or polyGlu). Expected outcomes include: 1) comprehensive exploration of composition diagrams, to characterize transitions (coacervate, gel, glass, aggregation/fibrillation) and their reversibility, via measurements of viscosity (particle tracking), diffusivity (FRAP, dynamic differential microscopy), along with visco-elastic measurements on microdroplets, 2) validation of challenging models of rigidification (e.g., gradual slowing down vs percolation of protein clusters) assessed by visco-elasticity and optical imaging of clusters during drying, 3) instrumental developments: microdialysis parallelized to screen either samples compositions or complex sequences of equilibration with buffers ; the rheometer fitted with high sensitivity load-sensor (to measure modulus down to 100 Pa on microdrops) & plate geometry expanding possibilities of liquid loading (to stop drying for long-lasting measurements, apply osmotic stresses) and eventually fitted with the in-situ dialysis microchannel. This final integration of in-situ dialysis should offer a broad range of possibilities for environment-triggered studies of the mechanical response of colloidal systems.