Fig. 2 | Nature Communications

Fig. 2

From: Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation

Fig. 2

UNC119 solubilises SFKs in a myristoyl-dependent manner. a, b FRET-FLIM of the interaction between either mCitrine fused to Src a or Fyn b and UNCA-mCh in HeLa cells (see also Supplementary Figs. 3 and 4). For each sample, the fluorescence intensity of SFK-mCit (donor), the fluorescence intensity of UNCA-mCh (acceptor), the spatial distribution of the mean fluorescence lifetime (τ) in nanoseconds and the molar fraction of interacting molecules (α) are shown per the false-colour look-up tables. The top rows depict the steady-state localisation of wild type (WT) SFK-mCit when co-expressed with UNCA-mCh. The second rows show representative measurements with the polybasic stretch mutant Src6Q-mCit a or non-palmitoylatable mutant FynC3S-C6S-mCit (b), and the bottom row shows the non-myristoylatable mutant SFKG2A-mCit proteins when co-expressed with UNCA-mCh. Bar graphs on the right depict average τ (c, e) and α-values (d, f) for different SFK proteins without (−), with UNCA-mCh (a), or UNCB-mCh co-expression (b; n = 10–20 cells/condition, data are mean ± SD from three independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by one-way analysis of variance (ANOVA) with Bonferroni post–hoc test). For α significance determination, samples were compared to the donor only control. Cells were sectioned into 10 segments of equal radius from the PM to the centre of the nucleus, and the mean intensity for each channel and α calculated for each segment (gj), for the Src-mCit profile angular masking was performed to look at the profile change across the perinuclear RE compartment. Line diagrams depict the normalised intensity for each channel (SFK-mCit in green and UNCA-mCh in magenta) and the mean α (black trace) in each segment ± SEM; the vertical dotted line indicates the average localisation of the nuclear membrane (n = 13 cells for Src-mCit, Src6Q-mCit and FynC3S-C6S-mCit, and n = 11 for Fyn-mCit). Scale bars, 10 μm

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