Lipid droplets as flexible substrates for protein phase separation.

Abstract

Biomolecular condensates have been shown to nucleate the formation of amyloid fibrils for many proteins implicated in neurodegenerative disorders, such as alpha synuclein in Parkinson's disease and fused in sarcoma (FUS) in amyotrophic lateral sclerosis. Within these fibrils, a distinct lipid signal is often observable, and indeed, a membrane environment is favorable for the formation of amyloid fibrils for a variety of biophysical reasons, including an increase in local protein concentration and conformational shift upon membrane binding. How might intracellular lipid substrates contribute to nucleation of phase separation? Here we observe for the first time that intrinsically disordered proteins can undergo phase separation on the surface of lipid droplets. We find that the low complexity domain of FUS (FUSLC) separates into protein-rich and protein-depleted phases at physiological salt (150mM) and protein concentrations (<5μM) when recruited to the surfaces of lipid droplets through a histidine-Ni-NTA headgroup interaction. The number of LDs showing FUSLC phase separation is strongly affected by variations in the protein and salt concentration, and it follows the trend seen previously with FUSLC droplets in solution. Further, these phase-separated patches demonstrate molecular mobility when assessed using fluorescence recovery after photobleaching (FRAP), which is indicative of a liquid-like state. This phenomenon extends to other well-known phase separating domains, such as LAF1-RGG. Overall, these results demonstrate the potential of monolayer substrates to nucleate protein phase separation and could have important implications for the assembly of intrinsically disordered lipid droplet-binding proteins.