Reconstitution of phase-separated signaling clusters and actin polymerization on supported lipid bilayers

Liquid–liquid phase separation driven by weak interactions between
multivalent molecules contributes to the cellular organization by
promoting the formation of biomolecular condensates. At membranes, phase
separation can promote the assembly of transmembrane proteins with their
cytoplasmic binding partners into micron-sized membrane-associated
condensates. For example, phase separation promotes clustering of nephrin,
a transmembrane adhesion molecule, resulting in increased Arp2/3
complex-dependent actin polymerization. In vitro reconstitution is a
powerful approach to understanding phase separation in biological systems.
With a bottom-up approach, we can determine the molecules necessary and
sufficient for phase separation, map the phase diagram by quantifying
de-mixing over a range of molecular concentrations, assess the material
properties of the condensed phase using fluorescence recovery after
photobleaching (FRAP), and even determine how phase separation impacts
downstream biochemical activity. Here, we describe a detailed protocol to
reconstitute nephrin clusters on supported lipid bilayers with purified
recombinant protein. We also describe how to measure Arp2/3
complex-dependent actin polymerization on bilayers using fluorescence
microscopy. These different protocols can be performed independently or
combined as needed. These general techniques can be applied to
reconstitute and study phase-separated signaling clusters of many
different receptors or to generally understand how actin polymerization is
regulated at membranes.