UC Berkeley

Biomolecular condensation has been recognized for the last decade-and-a-half as a ubiquitous modality of high-order molecular organization in living cells. This allows for compartmentalized, altered biochemistry that enables otherwise rare biochemistry. Condensates have been observed with both cytosolic (solution), and membrane-bound (surface) components. Linker for Activation of T Cells (LAT) and Epidermal Growth Factor Receptor (EGFR) are examples of transmembrane proteins that can undergo the latter type of condensation. The phase transition of the cytosol-facing phosphotyrosine tails of both of these proteins has been previously reconstituted on supported lipid bilayers (SLB). This type of phase transition is anabled by multivalent binding with downstream adaptors and signaling enzymes to form an extended, two-dimensional bond network. For LAT and EGFR, the adaptor protein Grb2 and the proline-rich scaffolding domain of the guanine exchange factor SOS, together, can reconstitute condensation. Previous work on these signaling condensates have shown that the condensed phase can directly impact downstream signaling kinetics. Additionally, the valency of crosslinking bonds and the bond association and dissociation kinetics within the condensate critically determine the phase transition kinetics and material properties of the resulting condensate. This dissertation details reconstitution of these condensates on SLBs along with fluorescence microscopy to further explore these faucets of signaling condensates. We find that condensation can be driven solely by binding to Grb2 which can dimerize to crosslink signaling proteins without SOS. This dimer interface can in turn be controlled through a phosphotyrosine on Grb2 itself to regulate condensation. This Grb2-mediated condensate retains its enhancement of Ras activation kinetics through SOS which can now be modulated through phosphorylation of Grb2. We also observe that we can reconstitute simultaneous condensates in solution and on the membrane with the same components. The solution condensates, on contact with the membrane surface decorated with a membrane condensate, can undergo a partial transition from a bulk three dimensional condensate to a surface-bound two dimensional condensate.