Advanced fluorescence microscopy techniques for the biophysical understanding of protein condensates
Department of Biomedical Engineering, University of California Irvine, CA, USA
Cells can organize many of their biochemical reactions in membrane-less compartments. These can be achieved by self-assembly of proteins leading to protein phase separations (PPS). The mechanism of de-mixing can be physiological (inner centromere) and/or pathological (stress granules). Quite often same biopolymer can drive different types of phase separation (liquid-like, gel-like, solid-like). From a biophysics perspective protein phase separation is a liquid de-mixing process where water/biomolecules interactions can affect the formation and the property of the phase separation. To characterize that, investigations commonly begin with the definition of the phase separation diagram of the purified protein that drive the water/protein liquid de-mixing. In our opinion, to better understand these complex phases, the characterization of the water dynamics is fundamental. Here, we proposed the study of the water dipolar relaxation measuring the emission spectra and fluorescence lifetime at optical resolution of ACDAN (6-acetyl-2-dimethylaminonaphthalene, solvathocromic dye) that colocalize on a model PPS. Indeed, we implement the method characterizing the phase diagram of a model PPS obtained by the coacervation of bovine serum albumin (BSA) in several ratios with a crosslinker and protein concentration. Here, phasor analysis of the ACDAN optical properties (fluorescence lifetime and emission spectrum) allowed to highlight differences in terms of water dynamics of the BSA coacervates even if the shape and size of the PPS were similar. This method can improve the definition of the phase diagram in solution and in cellular environments. We believe this approach can be useful to track the inner dynamics of the PPS especially when they can evolve to severe pathological conditions.