Membrane surfaces regulate assembly of a ribonucleoprotein condensate

Abstract

Biomolecular condensates organize biochemistry in time and space, yet little is known about how cells control either the position or scale of these assemblies. In cells, condensates often appear as dispersed, relatively small assemblies that do not grow (coarsen) into a single droplet despite their propensity to coalesce. Here we report that ribonucleoprotein condensates of the Q-rich protein Whi3 interact with the endoplasmic reticulum, prompting us to hypothesize that membrane association controls the position and size of condensates. Reconstitution of Whi3 condensates on supported lipid bilayers reveals that association with a diffusive lipid surface promotes condensation at both physiological ionic strength and protein concentration. Notably, these assemblies rapidly arrest, matching size distributions seen in cells. The timing of the arrest is influenced by the ordering of protein-protein and protein-RNA interactions and controlled by the slow diffusion of complexes induced by the membrane. This slowed diffusion limits both transfer of small protein-RNA complexes between condensates and their coalescence, thus driving coarsening to arrest. Our experiments reveal a tradeoff between locally-enhanced protein concentration at membranes, which favors condensation, and an accompanying reduction in diffusion, which restricts coarsening. Thus, membranes can maintain a population of small condensates in the absence of active mechanisms. Given that many condensates are bound to endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.One sentence summaryAssembly on a membrane surface positions and scales biomolecular condensates by controlling relative diffusion rates of proteins and nucleic acids.

Synergistic phase separation of two pathways promotes integrin clustering and nascent adhesion formation

“…Even with these physiological protein concentrations, we observe a significant increase in droplet density at 5 min and 10 min timepoints on bilayers containing integrin ( Figure 4g ). Thus, the β1 integrin cytoplasmic tail significantly increases the rate of condensate formation on bilayers, likely by increasing the local protein concentration at the membrane and thus accelerating condensate nucleation ( Snead et al, 2021 ).…”

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

“…Condensates, or clusters, of biomolecules not only form in the cytoplasm, but also on two-dimensional membrane surfaces [8][9][10][11][12] . This occurs when at least one of the species involved is embedded in the membrane: for instance, the clustering of transmembrane protein LAT activates signalling to produce cytokines when Tcell receptors are exposed to an antigen 9 , while protein Whi3 in fungi condenses with RNA on the endoplasmic reticulum 10 .…”

“…Predictions often rely on classical kinetic theory [29][30][31][32][33][34] , oblivious of biological systems' specificity, and whose validity needs to be tested on a caseby-case basis. In a dynamic environment such as that of a cell, where thermodynamic equilibrium is rarely reached, a better understanding of aggregation kinetics could provide new tools to extract information on the microscopic components from macroscopic experimental observations 10,[35][36][37] .…”

Linking transport and translation of mRNAs with endosomes and mitochondria

“…This assumption is supported by recent findings demonstrating that the fungal RNA‐binding protein Whi3 from Ashbya gossypii containing glutamine‐rich disordered regions forms biomolecular condensates associated with ER membranes. Importantly, membrane association of Whi3 controls the size of RNP condensates suggesting that interaction with lipid bilayers is crucial for regulating the precise formation and composition of these condensates in vivo (preprint: Snead et al , 2021). Similarly, Upa2 and Grp1 containing intrinsically disordered regions might influence formation of such RNP condensates at endosomal surfaces.…”