Journal: Science Advances

Article Title: A phospho-tyrosine–based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity

doi: 10.1126/sciadv.abq0084

Figure Lengend Snippet: ( A and B ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. The SBM in CSK and its mutation in CSK-AAA are indicated. ( C ) FA competition binding assay. Peptides containing the candidate SBM of CSK and the mutated sequence CSK-AAA compete with fluorescein-Puc 91–106 for binding to the SPOP MATH domain. [L] is the peptide concentration. Continuous lines are nonlinear least-squares fits to a complete competitive binding model. See for K d values. ( D ) Clustal Omega–based sequence alignment of Src family kinases and CSK. The SBM and the conserved tyrosine are highlighted by green and red boxes, respectively. ( E ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. ( F ) Nuclear translocation assay of SPOP-deficient RAW264.7 cells that were reconstituted with indicated forms of SPOP. ( G ) qPCR analysis of cells described in (F) that were stimulated with CpG-DNA for 4 hours. ( H to K ) In vitro kinase assays based on proteins expressed in HEK293T cells. CSK-AAA, SST340/341/341AAA. Fold differences of CSK autophosphorylation as determined by phosphor image analysis. The activity used for reference (set as 1) is underlined. Data represent mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.005 are determined by two-way ANOVA with Sidak’s multiple comparison test (G).

Article Snippet: N-terminal FLAG-tagged mouse CSK-WT and CSK-SST340/341/342AAA were expressed using the vector pCMV3-N-FLAG (Sino Biological). pcDNA3-N-FLAG-hIRF8 was from Sino Biological.

Techniques: Mutagenesis, Binding Assay, Sequencing, Concentration Assay, Nuclear Translocation Assay, In Vitro, Activity Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 Log 10 [D] (µm 2 s −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 µm 2 s −1 . I ( i – iii ) Examples of average mean-square displacement (MSD; µm 2 ) 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Transfection, Diffusion-based Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 (µm 2 ) and E , G area under the curve (AUC) of the corresponding MSD (µm 2 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 (µm 2 ) and L AUC of the MSD (µm 2 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Diffusion-based Assay, Western Blot, Transfection, Activity Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 (µm 2 ) curves over time (0.14 s). E Corresponding AUC (µm 2 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 (µm 2 ) curves over time (0.14 s). J Corresponding AUC (µm 2 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Diffusion-based Assay, Transfection, Binding Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 (µm 2 ) curves over time (0.14 s). M Corresponding AUC (µm 2 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Transfection, Diffusion-based Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 (µm 2 ). E Cluster lifetime (s). F Cluster area (µm 2 ). G Cluster membership (# trajectories/cluster). H Density within clusters (# detections/µm 2 ). 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 (µm 2 ). O Cluster lifetime (s). P Cluster area (µm 2 ). Q Cluster membership (# trajectories/cluster). R Density within clusters (# detections/µm 2 ). 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Diffusion-based Assay

Journal: Molecular Psychiatry

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

doi: 10.1038/s41380-022-01825-y

Figure Lengend Snippet: 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 (µm 2 ) curves over time (0.14 s). D Corresponding AUC (µm 2 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 (µm 2 ) curves over time (0.14 s). H Corresponding AUC (µm 2 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/μm 2 ) 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).

Article Snippet: The following constructs were used for neuronal transfections: pEGFP-N1 (Clontech #6085‐1), mCardinal (Addgene #54590), Fyn-mEos2, Fyn-Y531F-mEos2, Fyn-Y420F-mEos2 (Fyn-mEos2 containing the Y420F mutation), Fyn-K299M-mEos2 (Fyn-mEos2 containing the K299M mutation), Fyn-Y531F-K299M-mEos2, Fyn-ΔSH3-mEos2 (Fyn-mEos2 lacking the SH3 domain, amino-acids 82–143), Tau-P301L-GFP [ ] (human tau 2N4R, htau40, containing the FTLD mutation P301L, fused with EGFP at the C-terminus), Δtau74-GFP [ ] (human tau 2N4R lacking the MTBR and the C-terminal region, amino-acids 256–441, fused to green fluorescent protein (GFP)).

Techniques: Diffusion-based Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Activity Assay