Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes

Abstract

LAT is a membrane-linked scaffold protein that undergoes a phase transition to form a two-dimensional protein condensate on the membrane during T cell activation. Governed by tyrosine phosphorylation, LAT recruits various proteins that ultimately enable condensation through a percolation network of discrete and selective protein-protein interactions. Here we describe detailed kinetic measurements of the phase transition, along with coarse-grained model simulations, that reveal LAT condensation is kinetically frustrated by the availability of bonds to form the network. Unlike typical miscibility transitions in which compact domains may coexist at equilibrium, the LAT condensates are dynamically arrested in extended states, kinetically trapped out of equilibrium. Modeling identifies the structural basis for this kinetic arrest as the formation of spindle arrangements, favored by limited multivalent binding interactions along the flexible, intrinsically disordered LAT protein. These results reveal how local factors controlling the kinetics of LAT condensation enable formation of different, stable condensates, which may ultimately coexist within the cell.

Controlling cluster size in 2D phase-separating binary mixtures with specific interactions

“…However, re-entrant behavior of the dense phase with respect to the concentration has been observed in several phase-separating systems, suggesting that increasing the concentration of a given species does not necessarily promote the formation of large assemblies. While, in some cases, there appears to be an electrostatic-related change in the interactions, [14][15][16] in others, the effect seems to be fully stoichiometric [17][18][19][20] and most likely functional. 21 Computational work on biological condensates has built upon various models, ranging from simple lattice models 22,23 to offlattice coarse-grained polymers, 24,25 stickers and spacers, 26,27 or patchy colloids.…”

Controlling cluster size in 2D phase-separating binary mixtures with specific interactions

“…However, re-entrant behaviour of the dense phase with respect to concentration has been observed in sev-eral phase-separating systems, suggesting that increasing concentration of a given species does not necessarily promote the formation of large assemblies. While in some cases there appears to be an electrostatic-related change in the interactions [13][14][15] , in others the effect seems to be fully stoichiometric [16][17][18][19] and most likely functional 20 .…”

“…Computational work on biological condensates has built upon various models, ranging from simple lattice models 21,22 , to off-lattice coarse-grained polymers 23,24 , stickers and spacers 25,26 , or patchy colloids [17][18][19]22,27 . In many studies, though, self-assembly is represented within the scaffold-client context, which assumes the existence of a set of proteins (scaffold) that can alone phase-separate through homotypic interactions 2,19,27,28 .…”

A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

“…We reconstitute the cytoplasmic tails of EGFR on supported bilayers and characterize the system behavior upon interaction with Grb2 and SOS using total internal reflection fluorescence (TIRF) imaging. This experimental platform has been highly effective revealing both phase transition characteristics and functional signaling aspects of LAT protein condensates (4,5,10,(29)(30)(31). Published reports on the LAT system to date have emphasized SOS (or the SOS PR domain) as a critical crosslinking element.…”