In vitro experiments with engineered proteins: one composed of repeats of a single SH3 domain (SH3m, where m = 1–5), and the other composed of repeats of a PRM ligand (PRMn, where n = 1–5) showed change in optical properties (turbidity) due to the formation liquid droplets by phase separation as assessed by microscopy at high protein concentrations (change in protein concentration). The proteins were concentrated by about 100-fold in the droplets relative to the bulk phase. Higher valency (mutation) allowed for the formation of larger species (particle size and count) at a lower fractional saturation of the binding modules. The phase transition could be blocked by a high-affinity monovalent ligand. The multivalent proteins formed large polymers within the droplets (DLS, SAXS), such that the phase transition probably coincides with a sol–gel transition. The photobleaching recovery rate (FRAP) correlated inversely with the monomer–monomer affinity and valency, suggesting that recovery represents reorganization of a polymer matrix. The coexpression of mCherry–SH35 and eGFP–PRM5 fusion proteins in HeLa cells resulted in the formation of approximately 0.5–2-µm diameter (particle size and count) cytoplasmic (protein localization) puncta containing both fluorophores (protein co-localization) in vivo. The puncta did not stain with a large range of vesicle markers or a lipid dye, suggesting that they are phase-separated bodies rather than vesicular structures (morphology). The addition of NCK to an N-WASP construct caused droplet formation, as occurred in the model systems described above. The addition of a diphosphorylated (2pTyr) nephrin tail peptide dropped the phase boundary for both proteins by more than or equal to twofold (protein phosphorylation). This effect was even more pronounced when nephrin–3pTyr peptide (protein phosphorylation) was added (to the same total pTyr concentration), showing the importance of valency of the components and that the whole system could be regulated by kinases and phosphatases in vivo (PMID:22398450). Fluorescently tagged p-Nephrin, Nck and N-WASP co-localized to clusters formed on fluid supported lipid bilayers. Addition of 10 µM of a monovalent pTyr peptide derived from TIR (with KD of 40 nM for the Nck SH2 domain) to clusters formed from p-Nephrin /(SH3)3/N-WASP dissolved the clusters (particle size and count). Fluorescently tagged p-Nephrin (2200 molecules/µm²) was clustered by addition of 2 μM N-WASP and 1 μM Nck, addition of 10 nM Arp2/3 complex and 1 µM actin (10% rhodamine labeled) showed that actin specifically assembles on p-Nephrin/Nck/N-WASP clusters in an Arp2/3 dependent manner (protein co-localization) (PMID:25321392).