Fyn nanoclustering requires switching to an open conformation and is enhanced by FTLD-Tau biomolecular condensates

Abstract

Fyn is a Src kinase that controls critical signalling cascades and has been implicated in learning and memory. Postsynaptic enrichment of Fyn underpins synaptotoxicity in dementias such as Alzheimer's disease and frontotemporal lobar degeneration with Tau pathology (FTLD-Tau). The FLTD P301L mutant Tau is associated with a higher propensity to undergo liquid-liquid phase separation (LLPS) and form biomolecular condensates. Expression of P301L mutant Tau promotes aberrant trapping of Fyn in nanoclusters within hippocampal dendrites by an unknown mechanism. Here, we used single-particle tracking photoactivated localisation microscopy to demonstrate that the opening of Fyn into its primed conformation promotes its nanoclustering in dendrites leading to increased Fyn/ERK/S6 downstream signalling. Preventing the auto-inhibitory closed conformation of Fyn through phospho-inhibition or through perturbation of its SH3 domain increased Fyn's nanoscale trapping, whereas inhibition of the catalytic domain had no impact. By combining pharmacological and genetic approaches, we demonstrate that P301L Tau enhanced both Fyn nanoclustering and Fyn/ERK/S6 signalling via its ability to form biomolecular condensates. Together, our findings demonstrate that Fyn alternates between a closed and an open conformation, the latter being enzymatically active and clustered. Furthermore, pathogenic immobilisation of Fyn relies on the ability of P301L Tau to form biomolecular condensates, thus highlighting the critical importance of LLPS in controlling nanoclustering and downstream intracellular signalling events.

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).

Fig. 2. Dephosphorylation of the Y531 epitope controls the lateral entrapment of Fyn-mEos2, leading to downstream activation of ERK/S6 signalling.

A–C Representative intensity and diffusion coefficient maps for A Fyn-mEos2, B Fyn-Y531F-mEos2 and C Fyn-Y531F-K299M-mEos2 within spines of hippocampal neurons. Note that cooler colours within the intensity and the diffusion coefficient maps designate regions of higher localisation intensities and mobility, respectively. The surface of the spines is outlined for visibility. D–G Mobility of Fyn-mEos2, Fyn-Y531F-mEos2 and Fyn-Y531F-K299M-mEos2 in D, E dendrites and F, G spines, shown as D, F the MSD (µm²) and E, G area under the curve (AUC) of the corresponding MSD (µm² s). H–J Diffusion coefficient maps of H Fyn-mEos2, I Fyn-Y531F-mEos2 and J Fyn-Y531F-K299M-mEos2 expressed in HEK-293T cells. Boxed outlines are shown magnified below in (i). Note that hotter colours within diffusion coefficient maps designate regions of lower mobility. Corresponding K MSD curve (µm²) and L AUC of the MSD (µm² s). M Western blot of HEK-293T cells transfected with either mEos2 (empty control), Fyn-mEos2, Fyn-Y531F-mEos2 or Fyn-Y531F-K299M-mEos2. N Analysis of ERK1/2 activity and O S6 activity measured using the relative intensity of the corresponding western blot bands. Error bars are standard errors of the mean (SEM). Mean ± SEM values in D–G were obtained from neurons co-transfected with mCardinal and Fyn-mEos2 (N = 8), Fyn-Y531F-mEos2 (N = 14) or Y531F-K299M-mEos2 (N = 9). Mean ± SEM values in K, L were obtained from HEK-293T cells transfected with Fyn-mEos2 (N = 10), Fyn-Y531F-mEos2 (N = 11) or Y531F-K299M-mEos2 (N = 13). Mean ± SEM values in N, O were obtained from N = 3. Statistical comparisons were performed using a one-way ANOVA and Dunnett T3 test for multiple comparisons between groups in E, G and L; or were performed using a one-way ANOVA and Tukey’s test for multiple comparisons between groups in N and O. The specific adjusted p values accounting for multiple comparisons are reported for data considered significantly different (p < 0.05).

Fig. 3. Fyn is organised into nanoclusters in dendritic spines of hippocampal neurons and in HEK-293T cells.

A Representative image obtained after analysing the spatiotemporal distribution of Fyn-mEos2 trajectories and nanoclusters in hippocampal neurons using NASTIC. B, C Representative images plotting individual trajectories and clusters or individual trajectories coloured based on their instant diffusion coefficients ([D], with more immobile trajectories depicted in light colours, and more mobile trajectories depicted in dark colours), from B Fyn-mEos2 or C Fyn-Y531F-mEos2 in dendritic spines. D MSD of clustered trajectories (µm²). E Cluster lifetime (s). F Cluster area (µm²). G Cluster membership (# trajectories/cluster). H Density within clusters (# detections/µm²). I Representative image of Fyn-mEos2 epifluorescence in HEK-293T cells and the corresponding presentation of the spatiotemporal distribution of Fyn-mEos2 trajectories and their nanoclusters. Colour-coding of the clusters is based on their appearance in time across the acquisition (16,000 frames, 320 s). J Detail of an area (i) containing Fyn-mEos2 trajectories organised in multiple nanoclusters. K 3D (X, Y, Time) plot of Fyn-mEos2 trajectories from the region (i) in (J). Squares in X and Y represent 100 nm; squares in Time represent 20 s. L Representative intensity map of Fyn-mEos2 in a nanocluster of a HEK-293T cell. M Representative intensity map of Fyn-Y531F-mEos2 in a nanocluster of a HEK-293T cell. N MSD of clustered trajectories (µm²). O Cluster lifetime (s). P Cluster area (µm²). Q Cluster membership (# trajectories/cluster). R Density within clusters (# detections/µm²). Error bars are standard errors of the mean (SEM). Mean ± SEM values in D–H were obtained from NASTIC analysis of Fyn-mEos2 trajectories (N = 1495), and Fyn-Y531F-mEos2 trajectories (N = 962) from hippocampal neurons. Mean ± SEM values in N–R were obtained from NASTIC analysis of Fyn-mEos2 trajectories (N = 18,103), Fyn-Y531F-mEos2 (N = 20,434) from HEK-293T cells. Statistical comparisons in D–H and N–R were performed using unpaired Welch’s t-test. The specific adjusted p values accounting for the comparisons are reported when the data are considered significantly different (p < 0.05).

Fig. 4. Alteration of the SH3 domain promotes the lateral trapping of Fyn-mEos2.

A, B Representative intensity and diffusion coefficient maps of A Fyn-mEos2 or B Fyn-ΔSH3-mEos2 in dendrites of hippocampal neurons (DIV18-22). C Schematic representation of the tertiary structure of mEos2-tagged Fyn lacking its SH3 domain (Fyn-ΔSH3-mEos2), with a comparison of the domains of full-length Fyn and Fyn-ΔSH3 shown below. D Mobility of Fyn-mEos2 and Fyn-ΔSH3-mEos2 indicated as the MSD (µm²) curves over time (0.14 s). E Corresponding AUC (µm² s) of the MSD graph in D. F Representative image of Fyn-GFP epifluorescence in HEK-293T cells co-transfected with an mEos2-tagged anti-GFP nanobody (NB-anti-GFP-mEos2). The white box outline is shown at a higher magnification on the right to show individual NB-anti-GFP-mEos2 trajectories. G Representative image of Fyn-GFP epifluorescence in HEK-293T cells co-transfected with an mEos2-tagged anti-SH3 monobody (MB-anti-SH3-mEos2). The white box outline is shown at a higher magnification on the right to show individual MB-anti-SH3-mEos2 trajectories. H Schematic representation of the binding of anti-GFP-mEos2 nanobodies to the GFP of Fyn-GFP, and anti-Fyn-mEos2 monobodies to the SH3 domain of Fyn-GFP. I Mobility of Fyn-mEos2, NB-anti-GFP-mEos2 bound to Fyn-GFP, and MB-anti-SH3-mEos2 bound to Fyn-GFP indicated as the MSD (µm²) curves over time (0.14 s). J Corresponding AUC (µm² s) of the graphs in I. Error bars are standard errors of the mean (SEM). Mean ± SEM values in D, E were obtained from hippocampal neurons transfected with mCardinal and Fyn-mEos2 (N = 19) or Fyn-ΔSH3-mEos2 (N = 13). Mean ± SEM values in I, J were obtained from HEK-293T cells transfected with Fyn-mEos2 (N = 11), Fyn-GFP and NB-anti-GFP-mEos2 (N = 7), or Fyn-GFP and MB-anti-SH3-mEos2 (N = 7). Statistical comparisons were performed using Welch’s t-test in E, and one-way ANOVA and Tukey’s test for multiple comparisons between groups in J. The specific adjusted p values accounting for multiple comparisons are reported when the data are considered significantly different (p < 0.05).

Fig. 5. The FTLD P301L mutant Tau promotes the lateral trapping of Fyn-mEos2 in neurons and HEK-293T cells, altering the downstream ERK/S6 signalling.

A Representative epifluorescence image of GFP co-expressed in hippocampal dendrites with Fyn-mEos2. The white box outline is shown at a higher magnification below to display the intensity map of Fyn-mEos2. B Representative epifluorescence image of Tau-P301L-GFP co-expressed in hippocampal dendrites with Fyn-mEos2. The white box outline is shown at a higher magnification below to display the intensity map of Fyn-mEos2. C Mobility of Fyn-mEos2 co-expressed with GFP or with Tau-P301L-GFP in hippocampal neurons indicated as the MSD (µm²) curves over time (0.14 s). D Corresponding AUC (µm² s) of the graphs in C. E, F Representative diffusion coefficient maps of Fyn-mEos2 co-expressed with E GFP, or F Tau-P301L-GFP in HEK-293T cells. White boxed outlines are shown magnified on the right in (i). Note that hotter colours within diffusion coefficient maps designate regions of lower mobility. G Mobility of Fyn-mEos2 co-expressed with GFP or with Tau-P301L-GFP in HEK-293T cells indicated as the MSD (µm²) curves over time (0.14 s). H Corresponding AUC (µm² s) of the graphs in G. I Quantification of the % of clustered Fyn-mEos2 trajectories upon co-expression with GFP or with Tau-p301L-GFP. J Quantification of the density of Fyn-mEos2 clusters (clusters/μm²) upon co-expression with GFP or with Tau-p301L-GFP. K Western blot of HEK-293T cells transfected with either Fyn-myc and an empty vector, Tau-P301L-V5 and an empty vector, or Fyn-myc and Tau-P301L-V5. L Analysis of Tau phosphorylation at the Y18 epitope (pTau/Tau), M ERK1/2 activity (pERK/ERK) and N S6 activity (pS6/S6) measured using the relative intensity of the corresponding western blot bands. Error bars are standard errors of the mean (SEM). Mean ± SEM values in C, D were obtained from hippocampal neurons transfected with Fyn-mEos2 and GFP (N = 15) or Tau-P301L-GFP (N = 15). Mean ± SEM values in G–J were obtained from HEK-293T cells transfected with Fyn-mEos2 and GFP (N = 10 in G, H and N = 13 in I, J) or Tau-P301L-GFP (N = 9 in G, H and N = 22 in I, J). Mean ± SEM values in L–N were obtained from N = 3. Statistical comparisons in D, H, I and J were performed using unpaired Welch’s t-test. Statistical comparisons in L–N were performed using a one-way ANOVA and Tukey’s test for multiple comparisons between groups. The specific adjusted p values accounting for multiple comparisons are reported when the data are considered significantly different (p < 0.05).

Fig. 6. FRAP analysis of FTLD P301L mutant Tau shows slow recovery, but the deletion of the MTBD increases Tau mobility and reverts Fyn-mEos2 lateral trapping in neurons.

A Representative image of a hippocampal neuron transfected with Tau-P301L-GFP that has an intracellular Tau droplet. White box outlines indicate the cytosolic region and droplet-containing region where FRAP was performed, shown at a higher magnification in B and C, respectively. B, C Series of images acquired during FRAP analysis (prebleach 0 s, postbleach 2.59 s, 3.62 s and 51.23 s) of Tau-P301L-GFP in B the cytosol or C droplet outlined in A. D Plot of Tau-P301L-GFP FRAP analysis in the cytosol and droplet, as indicated. E Representative image of a hippocampal neuron transfected with Tau-P301L-GFP. The white box outline denoted by i is shown magnified below in (i). F, G Series of images acquired during FRAP analysis (prebleach 0 s, postbleach 3.12 s, 4.17 s and 45.35 s) of F Tau-P301L-GFP or G ΔTau74-GFP within dendritic spines. H Average plot of FRAP analysis of Tau-P301L-GFP or ΔTau74-GFP in dendritic spines, as indicated. I Quantification of the mobile fraction of the FRAP curves in H. J Representative epifluorescence images of mCardinal co-expressed in hippocampal dendrites with Fyn-mEos2 and Tau-P301L-GFP. White box outlines are shown at a higher magnification on the right with Tau-P301L-GFP epifluorescence and corresponding Fyn-mEos2 diffusion coefficient maps shown. Note that hotter colours within the diffusion coefficient map designate regions of lower mobility. K Representative epifluorescence images of mCardinal co-expressed in hippocampal dendrites with Fyn-mEos2 and ΔTau74-GFP. White box outlines are shown at a higher magnification on the right with ΔTau74-GFP epifluorescence and corresponding Fyn-mEos2 diffusion coefficient maps shown. Note that hotter colours within the diffusion coefficient map designate regions of lower mobility. L Mobility of Fyn-mEos2 co-expressed with Tau-P301L-GFP or with ΔTau74-GFP in hippocampal neurons indicated as the MSD (µm²) curves over time (0.14 s). M Corresponding AUC (µm² s) of the graphs in L. Error bars are standard errors of the mean (SEM). Mean ± SEM values in H, I were obtained from hippocampal neurons transfected with Tau-P301L-GFP (N = 18) or ΔTau74-GFP (N = 12). Mean ± SEM values in L, M were obtained from hippocampal neurons co-transfected with mCardinal, Fyn-mEos2 and Tau-P301L-GFP (N = 13) or mCardinal, Fyn-mEos2 and ΔTau74-GFP (N = 14). Statistical comparison in I and M were performed using the unpaired Welch’s t-test. The specific adjusted p values accounting for multiple comparisons are reported when the data are considered significantly different (p < 0.05).