The Liquid State of RIM1α and RBP Condensates is Maintained by Lipids.

At the presynapse, RIM1α and RIM-binding protein (RBP) play a crucial role in regulating vesicle docking and priming. Emerging evidence suggests that these proteins are involved in the organization of the active zone where they cluster with membrane proteins and lipids, forming protein condensates driven by liquid-liquid phase separation (LLPS). While protein phase separation has been associated with cellular function, it has also been linked to various disorders, as liquid condensates can promote protein aggregation, leading to dysfunction. In this work, we investigated the phase behavior of RIM1α and RBP. We find that under physiological conditions and in the absence of crowding agents, RIM1α and RBP have the ability to spontaneously form biomolecular condensates. Moreover, these liquid condensates have the propensity to mature over time, resulting in a liquid-to-solid transition. Using a combination of fluorescence microscopy with biophysical techniques and characterization methods, we confirm that these solid aggregates are β-sheet-rich and fibrillar in nature. These observations not only add to the growing evidence that supports that RIM1α and RBP can phase separate, but we also show that these proteins can aggregate into fibrillar structures within condensates. Finally, we find that in the presence of lipid vesicles, this liquid-to-solid transition is suppressed, indicating the potential role that lipids play in maintaining the liquid state of RIM1α/RBP condensates. In the context of protein aggregation, these biophysical observations report on the mechanisms behind the phase separation and subsequent aggregation of RIM1α and RBP.