Principles of Client Enrichment in Multicomponent Biomolecular Condensates

TL;DR

The paper addresses how client recruitment and phosphorylation modulate composition and critical behavior in multicomponent biomolecular condensates. It combines reconstituted FAK–Cas–PXN condensates with Flory–Huggins mean-field theory, modeling phosphorylation as changing heterotypic couplings ($chi_{12}$) and/or effective chain length ($N_2$) and treating PXN as a client that renormalizes scaffold interactions; analytical results yield closed-form critical-point expressions. Experiments show PXN lowers the threshold for associative LLPS and shifts the dense-phase composition toward FAK while depleting Cas, validating the renormalization picture. The work provides design principles for dynamic control of condensate composition and criticality via multicomponent regulation, bridging thermodynamic theory and reconstituted systems and informing how membranes and nonequilibrium processes may couple to condensate behavior.

Abstract

Biomolecular condensates are commonly organized by a small number of scaffold molecules that drive phase separation together with client molecules that do not condense on their own but become selectively recruited into the dense phase. A central open question is how client recruitment feeds back on scaffold interactions to determine condensate composition. Here we address this problem in a reconstituted focal adhesion system composed of focal adhesion kinase (FAK) and phosphorylated p130Cas (Cas) as scaffolds and the adaptor protein paxillin (PXN) as a client. We show that both FAK phosphorylation and PXN recruitment produce a common compositional response in which FAK becomes enriched while Cas is depleted within the condensate. To interpret these observations, we develop two complementary theoretical descriptions. First, within a two-component Flory-Huggins framework, we show that phosphorylation can be captured by either strengthening heterotypic FAK-Cas interactions or increasing the effective number of interaction-relevant segments on FAK, both of which bias partitioning toward FAK-rich condensates. Second, we introduce a minimal three-component Flory-Huggins theory without an explicit solvent and map it onto an effective two-component description, demonstrating that client recruitment renormalizes homotypic and heterotypic scaffold interactions. Analytical predictions for the location of the critical point are tested in reconstituted multicomponent systems through PXN addition, showing that client recruitment alone tunes proximity to criticality and reshapes condensate composition. Together, our results reveal distinct yet convergent physical routes by which post-translational modification and client recruitment control scaffold composition in multicomponent condensates.

Figures (6)

• Figure 1: Schematic illustrating the effects of phosphorylation and client proteins on condensate composition and concentration. (a) Phosphorylated Cas (pCas) and FAK act as scaffolds, while PXN serves as a client; weak multivalent interactions are indicated by double-sided arrows. (b) Scaffolds and the client are represented by triangular symbols of different colors. Grey circles represent condensates composed of (left) dFAK-pCas; (middle) pFAK-pCas; (right) pFAK-pCas-PXN. It shows how the condensate composition changes upon FAK phosphorylation (middle) and FAK phosphorylation combined with client enrichment (right), compared to their absence (left).
• Figure 2: Fluorescence microscopy images of focal adhesion condensates formed via phase separation. Condensates were assembled using four distinct protein compositions by combining all components at a concentration of 1 $\mu$M in buffer containing 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM DTT, and 1 mg/mL BSA. Images were acquired on an epifluorescence microscope 20 minutes after mixing to allow the system to reach equilibrium. The four compositions tested were: dFAK$-$PXN (dephosphorylated FAK, 15% GFP-labeled; phosphorylated Cas, 5% Alexa647-labeled; Nck; and N-WASP), dFAK$+$PXN (same as dFAK$-$PXN with the addition of PXN), pFAK$-$PXN (phosphorylated FAK, 15% GFP-labeled; phosphorylated Cas, 5% Alexa647-labeled; Nck; and N-WASP), and pFAK$+$PXN (same as pFAK$-$PXN with the addition of PXN). (A) GFP channel images used to quantify FAK intensity within condensates. (B) Alexa647 channel images used to visualize Cas intensity. Panels A and B correspond to the same condensates. Scale bars are 5 $\mu$m. (C,D) Quantification of fluorescence intensity within condensates. Images were thresholded and condensates segmented using ImageJ. Fluorescence intensities (arbitrary units, a.u.) of FAK (GFP) and Cas (Alexa647) were measured within condensates, and mean condensate intensities were calculated for each image. Experiments were repeated three times with five images collected per replicate. Data are displayed as box plots. Fluorescence intensities of FAK and Cas are not quantitatively comparable due to the use of different fluorophores and camera exposure settings.
• Figure 3: The phase diagrams are constructed from the Flory–Huggins free energy (Eq. \ref{['eq:FH3']} with $M = 2$) using a convex-hull algorithm. Red dashed lines indicate tie lines connecting the dilute and dense phases prior to FAK phosphorylation, while black dash-dotted lines denote tie lines after phosphorylation. The red and black binodals correspond to ternary systems composed of dFAK–Cas–solvent and pFAK–Cas–solvent, respectively. Phosphorylation of FAK is assumed to (A) enhance the FAK–Cas interaction ($\chi_{12}$) or (B) increase both the number of stickers on FAK ($N_2$) and FAK–Cas interaction ($\chi_{12}$), while keeping the Cas–Cas ($\chi_{11}$) and FAK–FAK ($\chi_{22}$) interactions, as well as the number of stickers on Cas ($N_1$), constant. The parameters are shown in the legend, and the rest of the parameters used in the phase diagram construction are: $\chi_{11} = 2.8$, $\chi_{22} = 0.2$. The blue circle denotes $\phi_{\mathrm{Cas}} = 0.075$ and $\phi_{\mathrm{FAK}} = 0.096$, while the red and black circles denote the dense-phase compositions before and after phosphorylation of FAK, respectively. The circles show FAK phosphorylation is found to increase the FAK concentration while depleting Cas within the condensate; nevertheless, it renders the dense phase overall denser, as indicated by the larger area enclosed by the binodal.
• Figure 4: Theoretical prediction for the effect of the client PXN on Cas–FAK phase separation in an effective two-component Flory–Huggins framework. Phase diagrams are constructed using the convex hull method from the Flory-Huggins free energy (Eq. \ref{['eq:FH3']} for dFAK-pCas system and Eq. \ref{['eq:FH3nosolvnewparams']} for dFAK-pCas-PXN system), and plotted with the volume fractions of Cas (FAK), denoted by $\phi_{\text{Cas}}$ ($\phi_{\text{FAK}}$), along the horizontal (vertical) axes. Red solid binodal corresponds to dFAK–Cas mixture in the presence of an explicit solvent, whereas black solid binodal refers to the dFAK–Cas–PXN system without an additional solvent, with PXN effectively acting as the solvent. In dFAK–Cas mixture, interactions between the two scaffolds include both homotypic and heterotypic contributions, described by the Flory–Huggins parameters $\chi_{11}$, $\chi_{22}$, and $\chi_{12}$. Additionaly, dFAK–Cas-PXN mixture incorporates scaffold–client interactions, which modifies the scaffold–scaffold interactions and lead to effective homotypic and heterotypic interaction parameters for the scaffolds. The resulting effective interaction parameters are $\xi_{1} = \chi_{11} + \chi_{33} - 2\chi_{13}$, $\xi_{2} = \chi_{22} + \chi_{33} - 2\chi_{23}$, and $\xi_{3} = \chi_{12} + \chi_{33} - \chi_{13} - \chi_{23}$. The parameters used to construct the phase diagrams are as follows: (Red) $\chi_{11} = 2.8$, $\chi_{22} = 0.2$, $\chi_{12} = 3.5$; $N_{1} = 10$, $N_{2} = 6$; (Black) $\chi_{11} = 2.8$, $\chi_{22} = 0.2$, $\chi_{33} = 1.0$, $\chi_{12} = 5.2$, $\chi_{13} = 0.6$ and $\chi_{23} = 0.4$, $N_{1} = 10$; $N_{2} = 10$, which lead to the effective interaction parameters $\xi_{1} = 2.6$, $\xi_{2} = 0.4$, and $\xi_{3} = 5.2$. The blue circle denotes $\phi_{\mathrm{Cas}} = 0.075$ and $\phi_{\mathrm{FAK}} = 0.096$, while the red and black circles denote the dense-phase compositions before and after the addition of PXN, respectively.
• Figure 5: Effect of phosphorylation and client (PXN) addition on the phase diagrams of a system consisting of two scaffolds (FAK and Cas) and one client (PXN), with no solvent. Phase diagrams are constructed using Flory-Huggins free energy expressed in Eq. \ref{['eq:FH3']} and Eq. \ref{['eq:FH3nosolvnewparams']}. FAK phosphorylation effectively modifies the heterotypic scaffold–scaffold interactions ($\chi_{12}$) and FAK sticker numbers ($N_2$), while client addition also alters all effective homotypic and heterotypic interactions among the scaffolds. The effective Flory–Huggins interaction parameters ($\xi_{1}$, $\xi_{2}$, and $\xi_3$) between the scaffolds are defined in terms of the scaffold–scaffold and scaffold–client interaction parameters, as described after Eq. \ref{['eq:FH3nosolvnewparams']}. The actual and effective interaction parameters are as follows: (Red) $\chi_{11} = 2.8$, $\chi_{22} = 0.2$, $\chi_{33} = 1.0$, $\chi_{12} = 3.5$, $\chi_{13} = 0.6$$\chi_{23} = 0.4$, $N_{1} = 10$, and $N_{2} = 6$, yielding effective interactions $\xi_{1} =2.6$, $\xi_{2} = 0.4$ and $\xi_{3} = 3.5$; (Black) $\chi_{11} = 2.8$, $\chi_{22} = 0.2$, $\chi_{33} = 1.0$, $\chi_{12} = 5.2$, $\chi_{13} = 0.6$$\chi_{23} = 0.4$, $N_{1} = 10$, and $N_{2} = 10$, yielding effective interactions $\xi_{1} = 2.6$, $\xi_{2} = 0.4$ and $\xi_{3} = 5.2$. The blue circle denotes $\phi_{\mathrm{Cas}} = 0.085$ and $\phi_{\mathrm{FAK}} = 0.09$, and red and black circles denote the dense phase compositions before and after the addition of PXN and phosphorylation of FAK, respectively. The dense-phase is enriched in FAK and depleted in Cas upon phosphorylation and PXN addition.