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Characterization of the effects of relative and absolute valency on phase separation and partitioning

Multivalency is a common theme in biology from antibodies to cadherins to the EGFR. The ability of biology to strengthen or weaken interactions through the creation or destruction of multiple binding sites in a highly dynamic way is central to many cellular functions. Binding sites that can be “created” or “destroyed” by posttranslational modifications are of particular utility as they do not generally require the energy- and time-consuming process of transcription and translational in order to generate more binding sites. In effect, posttranslational modifications have the ability to increase the number of binding sites many fold without target protein levels changing at all. This multivalency phenomenon also allows for avidity and polymerization effects since the binding sites are more densely dispersed. The result is that these types of multivalent systems have the capacity to be very sharply and rapidly controlled, making them ideal for signaling cascades.

Our lab previously showed that proteins with multivalent, interacting subunits/motifs can polymerize and phase separate in vitro and in cells. This has been demonstrated in the context of several systems: SH3-PRM, Nephrin/Nck/N-WASP, and SUMO-SIM. I am studying two aspects of multivalency driven phase separation.

First, I am working to understand how phase separation, and the partitioning of markers into phase-separated liquid-like droplets, change as a function of relative valency of the polymerizing species (i.e. the ratio/difference of one binding motif, such as SUMO, to the other binding motif, in this case SIM). The goal is to determine how phase separation and partitioning can be affected by dynamic regulation of valency in vivo by modeling the valency combinations in vitro. Since cells can modulate SUMOylation in a highly dynamic manner, it is conceivable that partitioning of molecules into and out of a SUMO-SIM phase separated body could be rapidly controlled by changes in the SUMOylation machinery.  This, in turn could allow rapid control of biochemical processes that occur within these bodies.

Second, I am investigating the effects of recruitment to phase separated structures on the kinetics of an enzyme-substrate system, specifically SUMOylation. We hope to see if recruitment acts merely to increase the concentration of enzymes and substrates, or whether it imparts additional positive or negative pressure on enzyme kinetics. In essence the same process (SUMOylation) could control the formation, composition and function of phase separated bodies.  Taken together, this represents a potentially very dynamic and tunable system to control cellular processes.