Fig 2: Phase separation modulated by vesicle curvature and lipid composition.a A mixture of recombinant SLP65 with either SUVs (Rh ˜ 20 nm) or LUVs (Rh ˜ 60 nm) was fractionated by a sucrose gradient flotation assay, and the different layers were subjected to anti-SLP65 immunoblot analysis (apparent MW indicated in kDa). Vesicles will float to the top and are found in layers 1 and 2. Bar graph shows the percentage of SLP65 associated with vesicles based on the immunoblot signal intensity. b Confocal fluorescence microscopy of a mixture of different concentrations of Atto 430LS-tagged SLP651-330 and CIN85?57 together with SUVs containing DOPC:DOPE of 75:25 mol% (b, top) and DOPC:DOPE:DOPS:cholesterol of 35:25:10:30 mol% (b, bottom), respectively. (Scale bar = 10 µm) Source data are provided as a Source Data file. c SLP65-deficient DT40 B cells were transduced with constructs encoding citrine-tagged SLP65?N fusion proteins with the N-BAR domain of Amphiphysin. Wild-type SLP65 and SLP65?N-expressing cells served as positive and negative controls, respectively. The domain structures of the expressed SLP65 variants are depicted in the upper part of (c). Cells were loaded with the Ca2+-sensitive fluorophore Indo-1 and Ca2+ flux was monitored by flow cytometry. Cells were stimulated with anti-BCR antibody (M4) at the indicated time point. d Confocal fluorescence microscopy of cells transduced with N-BAR-SLP65?N were either left untreated (upper panel) or stimulated via their BCR (lower panel).

Fig 3: SLP65 interacts specifically with GEF-H1 and triggers its activation. (A) Left panel- Immunoprecipitation followed by western blot analysis for the interaction of endogenous SLP65 with GEF-H1 in DG75EB/HA-RhoA cells upon BCR stimulation. Cells were stimulated with anti-IgM antibody for the indicated time period and IP was carried out with anti-SLP65 antibody. PLC?2, a known interactor of SLP65 is used as a positive control. Result representative of 3 independent experiments. Right Panel- Quantification of immunoprecipitated GEF-H1 with respect to SLP65. Statistical analysis- One-way ANOVA. (B) Left- Proximity ligation assay (PLA) to detect the association of SLP65 and GEF-H1 in unstimulated DG75EB/HA-RhoA cell line. Only secondary antibodies are used in the controls. Close proximity is shown as red dots. Right- quantification of number of signals per cell, error bars represent mean ± SD. Unpaired t-test, two-tailed. (C) PLA to detect the association of SLP65 and GEF-H1 in murine B cells isolated from bone marrow (BM) and spleen (SP) and human peripheral blood (PBL) B cells. B cells are isolated by CD19 magnetic sorting. Close proximity in a range of 10-40 nm is shown as red dots. Right- quantification of number of signals per cell, error bars represent mean ± SD. (D) Left- western blot analysis for the detection of phosphorylated GEF-H1 upon SLP65 activation in bone marrow derived early pre-B cells from Rag2-/- , ?5-/- , Slp65-/- triple knock out (TKO) mice. The cells were reconstituted with µ-heavy chain, ?5 and inducible SLP65-ERT2 construct and then induced with 2µM 4-hydroxy tamoxifen (4-OHT) or ethanol (Et) for 30 min to activate SLP65. GAPDH is used as loading control. Right- quantification of pGEF-H1 band intensity with respect to GAPDH. Result representative of 4 independent experiments. Statistical analysis- unpaired t test, two tailed. *p<0.05, ****p<0.0001, ns, not significant.

Fig 4: Tripartite phase separation of SLP65 with CIN85 and SUVs.a Confocal fluorescence microscopy of SLP65-deficient DT40 B cells that had been reconstituted with a citrine fusion protein of either wild-type SLP65 (left), a variant lacking N-terminal 45 amino acids (?N, middle), or a CIN85 binding-deficient variant (?PRM, right). b Domain architecture of the different constructs of SLP65 and CIN85 studied here (PRR: proline-rich region, CC: coiled coil). c–e Confocal fluorescence microscopy of a mixture of different concentrations of Atto 430LS-tagged SLP651-330 with the corresponding equal concentrations of either (c) CIN851-333, (d) CIN85?57, or (e) CIN85?57 and SUVs made from 1 mM of phospholipids (see Method). The SUVs had a lipid composition of DOPC:DOPE:DOPS = 65:25:10 mol% and a mean hydrodynamic radius of 23 nm. Concentrations of CIN85?57 refer to the monomeric concentration. Index of dispersion of the fluorescence intensity were shown as box-and-whisker plots (12 ROIs and two independent samples, n = 24). The line inside the box and the edges of the box correspond to the median, first and third quartiles, respectively. The outliers with value =1.5 times the interquartile range away from the top or bottom of the box are denoted by circles. Source data are provided as a Source Data file. f Different concentrations of Atto 430LS-tagged SLP6540-330 were mixed with the corresponding equal concentrations of CIN85?57 together with 1 mM of SUVs. g Mixture of 5 µM of Atto 430LS-tagged SLP651-330 and 1 mM of SUVs without CIN85 (left) or with 5 µM of CIN85?57 (right). Scale bars equal to 10 µm (a, c–g).

Fig 5: SLP65 interacts with RHOA and regulates its activation. (A) Left- PLA showing the association of SLP65 and RHOA in DG75EB/HA-RhoA cell line. Only secondary antibodies are used in the controls. Close proximity is shown as red dots. Right- quantification of number of signals per cell, error bars represent mean ± SD. Unpaired t-test, two-tailed. (B) Left- PLA showing the association of RHOA with phosphorylated SLP65 in DG75EB/HA-RhoA cell line upon BCR stimulation. Cells were stimulated with anti-IgM F(ab)2 antibody for the specified time period. Right- quantification of number of signals per cell, bars represent mean ± SD. Unpaired t-test, two-sided. (C) Detection of total cellular RHOA activity by G-LISA RHOA activation assay. Reconstituted bone marrow derived pre B cells from TKO mice were treated with ethanol (control) or 4-OHT to induce SLP65 for the indicated time point. Equal amounts of cell lysates from control and 4-OHT treated cells were used for the assay. Statistical analysis: one sample t test. (D) Western blot analysis for the detection of RHOA upon SLP65 activation in TKO cells. The cells were reconstituted with µ-heavy chain, ?5 and inducible SLP65-ERT2 construct and then induced with 2µM 4-OHT or Et for 30 min to activate SLP65. GAPDH is used as loading control. *p<0.05, ***p< 0,001, ****p<0.0001, ns, not significant.