Fig. 1: Single-molecule tracking photoactivated localisation microscopy (sptPALM) of Fyn-mEos2.

A Illustration showing (i) the protein domains of Fyn (SH1-4 and PPII), with the boxed outline magnified below in (ii) to highlight key epitopes in the SH1 domain (K299 and Y420) and C-terminal tail (Y531) of Fyn. B Tertiary structure of Fyn in its closed, inactive conformation. mEos2 is conjugated to the C-terminus of Fyn. C Tertiary structure of Fyn in its open, active conformation. mEos2 is conjugated to the C-terminus of Fyn. D–G SptPALM of Fyn-mEos2 co-transfected with GFP in secondary dendritic branches and spines of hippocampal neurons (DIV19-22). Panels depict representative D GFP epifluorescence image, E localisation intensity map, F diffusion coefficient map, and G trajectory map for Fyn-mEos2. Note that cooler colours within the intensity and the diffusion coefficient maps in (E) and (F) designate regions of higher localisation intensities and mobility, respectively. Boxed outlines in the left panels are shown magnified on the right. H(i–iii) Examples of frequency distribution of Fyn-mEos2 diffusion coefficients [D] (plotted as Log₁₀ [D] (µm²s⁻¹)) from individual neurons. H(iv) Average Fyn-mEos2 frequency distribution of diffusion coefficients from (i), (ii) and (iii). The threshold separating immobile and mobile molecules (dotted line) was set at −1.6 µm²s⁻¹ [49]. I(i–iii) Examples of average mean-square displacement (MSD; µm²) curves over time (0.14 s) of trajectories from individual neurons. I(iv) Average Fyn-mEos2 MSD from (i), (ii) and (iii). Error bars are standard errors of the mean (SEM).