Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions

Abstract

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.

Figure 1

(A) Representative giant unilamellar vesicles (GUVs) showing temperature-dependent liquid-liquid phase separation. At 31°C, which is above the transition temperature T_misc of 29°C, the distribution of lipids is homogeneous across the membrane for 100% of the sample of ∼100 vesicles. Below T_misc at 24°C (right), lipids compartmentalize into macroscopic domains for 99% of the sample of ∼100 vesicles: the L_d domain (TR-DHPE; yellow) is enriched with unsaturated lipids, and the L_o with saturated lipids (OG-DHPE; blue). All GUV experiments are performed in buffer with matching osmolarity (50 mM Tris and 150 mM NaCl (pH 7.4)). Typical vesicle concentrations were ∼0.2 mg/mL. Scale bars, 5 μm. The ensemble average temperature-dependent phase separation (n ∼100 vesicles) is shown in Fig. 4 (right panel, empty circles). (B) In lipid raft theory, clusters of signaling proteins, such as the TCRs, are “carried” on ordered lipid domains to facilitate signal transduction.

Figure 2

The LAT:Grb2:SOS protein condensate was reconstituted on GUVs. His-tagged pLAT is associated with the vesicles by chelating to Ni-nitrilotriacetic-acid lipids. The introduction of a 1.2-μM, full-length Grb2 and 0.8 μM proline-rich domain of SOS results in extended networks of LAT condensate that is visualized by the AF555 fluorescence in the confocal microscopy (top). The LAT:Grb2:SOS assembly can be reversed by dephosphorylation of LAT by phosphatase (5 μM YopH) (bottom). Under the experimental conditions used, LAT showed condensation on most (>95%) of the vesicles and reversal by phosphatase (two independent experiments, n ∼50 vesicles). Scale bars, 5 μm. To see this figure in color, go online.

Figure 3

An example of a video showing the LAT:Grb2:SOS condensate-induced lipid phase separation on GUVs. Starting with a temperature (31°C) above its T_misc (29°C), the lipids are initially spatially homogeneous. As the proteins assembled (visualized by doping unlabeled Grb2 with 1% Grb2-AF647), the lipids undergo liquid-liquid phase transition. OG-DHPE and TR-DHPE mark the L_o and L_d regions, respectively. All vesicles that were capable of phase separation transitioned within 30 s. The ensemble average data for the temperature-dependent phase separation (n ∼100 vesicles) are shown in Fig. 4 (right panel, red circles). Scale bars, 5 μm. To see this figure in color, go online.

Figure 4

(Left) At 31°C, the GUVs associated with LAT:Grb2:SOS clusters are phase separated. Note that the smallest vesicle, invaginated within a larger vesicle, is inaccessible to the proteins and remains homogeneous. At 39°C, Grb2 has dissociated from the smaller vesicle, which became homogeneous. For the larger vesicle on which the protein condensate remains, the lipid of the phase separation also remains. Scale bars, 2 μm. (Right) The miscibility transition temperatures (T_miscs) were measured for bare GUVs (T_misc = 29.3 ± 0.5°C), GUVs with LAT (T_misc = 27.8 ± 0.4°C), and GUVs with the LAT condensate (T_misc = 33.9 ± 0.5°C) by counting the fraction of phase-separated vesicles as a function of temperature then fitting them to the logistic function. Although the difference in T_misc between bare GUVs and LAT-associated GUVs are minimal, it is increased significantly in the presence of the protein assembly. The data primarily reflect temperature-dependent LAT:Grb2:SOS interactions rather than GUV phase separation because the protein assembly becomes unstable at high temperatures and dissociates from the vesicles. However, hypothetically, stable LAT:Grb2:SOS interactions would further increase the apparent T_misc. The GUV counts are shown in Table S1. To see this figure in color, go online.

Figure 5

(A) The LAT:Grb2:SOS condensate on GUVs results in the segregation of K-Ras into the L_d region with the condensate, suggesting that spatial organization mediated by protein assemblies can propagate downstream of the signaling pathway via lipids. K-Ras templated the L_d region marked by TR on >95% of the vesicles (two independent experiments, n ∼ 30 vesicles) with the apparent partition coefficient $K=\left(Ra{s}_{in}/Ra{s}_{out}\right)\sim 4.7\pm 0.4$ (where ± denotes SEM). The temperature was 22°C, at which all of the vesicles were phase separated. Scale bars, 5 μm. (B) This lipid phase separation induced by protein organization may underlie lipid rafts seen in TCR clusters. To see this figure in color, go online.