Figure 3.

The formation of LLPS pFGFR2-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers and plasma membranes

(A) pFGFR2_Cyto-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers. (i) Confocal images of homogeneously distributed pFGFR2_Cyto Atto-488 (20 μM, 6xHis tagged) on membrane bilayers, (ii) pFGFR2_Cyto Atto-488 gradually clustered upon the addition of SHP2_C459S Atto-594 (60 μM, untagged), and (iii) pPLCγ1 Atto-647 (6 μM, untagged), followed by (iv) additional 36 μM of untagged pPLCγ1 Atto-647. Scale bars, 10 μm.

(B) FRAP analysis showing the dynamic nature of pFGFR2_Cyto-SHP2_C459S-pPLCγ1 condensates on supported lipid bilayers as all pFGFR2_Cyto, SHP2_C459S, and pPLCγ1 exchanged with their counterparts in the dilute phase. Data are presented as mean ± SD, n = 2 experiments.

(C) Immunofluorescence staining images showing colocalized SHP2-Alexa 488 and PLCγ1-Alexa 647 droplet formation on plasma membrane in FGF9-stimulated (10 ng/ml, 15 min) Caco-2 cells and Caco-2 FGFR2i cells. Inset image: magnification of regions shown to exemplify endogenous SHP2-PLCγ1 clusters on membranes. Graph (right of image): statistical analysis of droplet formation in parental Caco-2 cells and Caco-2 FGFR2i cells. Only the SHP2-Alexa 488 and PLCγ1-Alexa 647 colocalized droplets were counted. Knocking down FGFR2 reduces SHP2-Alexa 488 and PLCγ1-Alexa 647 colocalized droplets. Degree of colocalization of endogenous PLCγ1 and SHP2 determined by Pearson's R value. Sample numbers = 37 (wild type) or 38 (FGFR2i) from 2 independent experiments.

(D) Live cell images showing FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation on plasma membrane upon FGFR2_ΔVT expression and activation in HEK293T SHP2 KO cells. FGFR2_ΔVT, SHP2_C459, and PLCγ1 were tagged with Neptune 2.5, mOrange, and mEGFP, respectively. Alexa 350-conjugated wheat germ agglutinin was used to stain the plasma membrane. (i) Serum-starved (-FGF9) cells show a low level of FGFR2_ΔVT-SHP2_C459S-PLCγ1 droplets colocalized on membrane; this could have been due to protein recruitment by the basally activated FGFR2. Most SHP2 and PLCγ1 proteins are diffused in cytosol. (ii) FGF9-stimulation (+FGF9, 10 ng/ml for 15 min) led to the activation of FGFR2 and enhanced FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation on the plasma membrane (see also Video S1). (iii) Expression of fluorescent tags alone does not initiate the droplet formation. (iv) In the absence of SHP2_C459S-mOrange and PLCγ1-mEGFP, activated FGFR2_ΔVT still forms droplets on the membrane with other endogenous cellular proteins. (v) SHP2_C459S-mOrange does not form droplets in the absence of FGFR2_ΔVT. (vi) PLCγ1-mEGFP does not form droplets in the absence of FGFR2_ΔVT. (vii) FGFR2_ΔVT cannot recruit active PLCγ1-mEGFP to the membrane in the absence of SHP2 expression, resulting in the random diffusion of PLCγ1-mEGFP.

(E) Statistical analysis of FGFR2_ΔVT-SHP2_C459S-PLCγ1 LLPS droplet formation in the absence (light peach) or presence (dark peach) of FGF9 stimulation. Sample numbers = 40 per condition from 3 independent experiments. Data are presented as mean ± SD.