Fig. 2: Oligomerisation in vitro on reconstituted supported lipid bilayers.

a Cartoon depicting the experimental set-up of a supported lipid bilayer with tethered His-EGFP (visualised as green beta barrel inspired by PDB 1f0b¹⁰²). Oligomerisation was induced by use of primary (orange) and secondary antibodies (magenta). b Confocal images of SLBs composed of DOPC and DGS-Ni-NTA (4%) labelled with EGFP-His (left), treated with primary (mouse) anti-GFP antibody (middle), and addition of secondary antibody (anti-mouse, right). Scale bar is 5 µm. Representative images from >3 independent experiments. c BTS diagram for EGFP (left, control), EGFP and anti-GFP antibody (+Primary, middle), and EGFP, primary, and secondary antibody (+Primary/Secondary, right). All BTS histograms are significantly different from each other with p < 0.01 (permutation test). d Changes in numbers of particles per area as determined by sFCS. e Experimental fluctuation data quality measured by nRMSD and SNR as violin plots (dashed grey lines represent median and dotted lines represent quartile values). f Cartoon depicting the interaction of a membrane-anchored EGFP with a fluorescently tagged nanobody undergoing energy transfer (FRET) g BTS histograms for EGFP (control, left) and with anti-GFP nanobody (labelled with AbberiorSTAR635P, right), p < 0.01 (permutation test) h Cartoon of the reconstituted system for investigation of CD40 organisation. AlexaFluor488 conjugated to a His₆-tag (AF488-His) was used as a monomeric control (left). Recombinant CD40-His was labelled with AF488 (orange with green star). Perturbations performed with anti-CD40 antibodies (black) or recombinant CD40LG (red). i Confocal images of SLBs with varying amounts (measured by sFCS) of CD40-AF488-His. Scale bar is 5 µm. j BTS histogram for AF488-His on a SLB as monomeric control k sFCS BTS diagrams for different surface concentrations of CD40-AF488-His. 25 molecules/µm² is significantly different from 100 molecules/µm² (p < 0.01) whereas 175 molecules/µm² is not significantly different from 100 molecules/um² (p > 0.05, permutation test). l, m sFCS BTS diagram for CD40-AF488-His (monomeric control, l) and under perturbation (m) with anti-CD40 antibodies (5C3 and HB14) or recombinant CD40LG-His (all unlabelled). Conditions in m are significantly different from the control in n with p < 0.01 (permutation test). 5C3 is not significantly different from HB14. CD40L vs 5C3 p = 0.1 and CD40L vs HB14 p = 0.01 (permutation test). The experimental histograms are representative data from one repeat and integrate more than 10 sFCS acquisitions on various bilayer locations (>500 autocorrelation curves) acquired at ambient temperature (24 °C).