Measuring the phase behavior of synapsin in vitro

The phase separation of biopolymers has been recently found to be a means of intracellular spatial and temporal organization. However, there are still many species of biomolecular condensates for which the physical factors that drive phase separation have yet to be elucidated. Here, we aim to understand the interactions which impact the phase separation of synapsin 1a, which has been shown to phase separation via to interactions in its disordered C-terminus. Recent works have shown that the phase behavior of synapsin 1a is dependent on monovalent salt concentration and have shown that synapsin 1a condensate properties can be mediated by depletion agents and associated organelles and proteins that colocalize with synapsin within cells, such as α-Synuclein and synaptic vesicles. Here, we investigate the physical parameters that affect synapsin 1a phase separation by measuring the phase behavior of isolated synapsin 1a using confocal microscopy. We visualize fluorescently-tagged synapsin 1a that has been expressed in the Expi293 cell system and purified using nickel affinity chromatography and size exclusion chromatography and measure the in vitro phase diagram of synapsin 1a as a function of e.g., salt concentration, temperature, and size of depletants. We examine the critical concentrations and concentration-dependent effects of each parameter to uncover the biophysical interactions that dictate phase separation of synapsin 1a.