Cells are organized on length scales ranging from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. Here we show that interactions between diverse, synthetic multivalent macromolecules (including multi-domain proteins and RNA) produce sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its established biological partners Nck and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology.

SUMMARY Members of the Wiskott-Aldrich Syndrome Protein (WASP) family control actin dynamics in eukaryotic cells through stimulating the actin nucleating activity of the Arp2/3 complex. The prevailing paradigm for WASP regulation invokes allosteric relief of autoinhibition by diverse upstream activators. Here we demonstrate an additional level of regulation that is superimposed upon allostery: dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly by this system. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3 domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between different family members. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.