Journal: Nature Communications

Article Title: Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles

doi: 10.1038/s41467-023-43130-4

Figure Lengend Snippet: a SdTIM of VAMP2-pHluorin-bound At647N-GBP nanobodies in SVs, indicative of recycling pool SV mobility. (i) Epifluorescence image of a neuronal segment expressing VAMP2-pHluorin acquired before incubation with At647N-GBP. Inset (red outline) highlights a presynaptic compartment, shown at higher magnification in (ii). (iii) Fluorescence intensity, (iv) diffusion coefficient (the color bar represents log 10 [μm 2 s −1 ]) and (v) trajectory maps of recycling SVs. b SptPALM of vGLUT1-mEos2-containing vesicles, indicative of the total pool SV mobility. (i) Epifluorescence image of a neuronal segment expressing vGLUT1-mEos2. Inset (red outline) highlights a presynaptic compartment, shown at higher magnification in (ii). (iii) Fluorescence intensity, (iv) diffusion coefficient (the color bar represents log 10 [μm 2 s -1 ]) and (v) trajectory maps of total SVs. c , Average MSD of VAMP2-pHluorin/At647N-GBP trajectories (Recycling pool; black), and vGLUT1-mEos2 (Total pool; red) as a function of time. d Area under the MSD curve (AUC; µm 2 s). e Frequency distribution of the diffusion coefficients [D] shown in a semi-log plot. Grey dashed line indicates the threshold used to distinguish the immobile (Log 10 [D] ≤ −1.6) from the mobile (Log 10 [D] > −1.6) fraction of molecules. f Plot of the mobile fraction of molecules. g Three-state model of diffusive states inferred by vbSPT analysis of VAMP2-pHluorin/At647N-GBP trajectories. h , Three-state model of diffusive states inferred by vbSPT analysis of vGLUT1-mEos2 trajectories. Circles in ( g ) and ( h ) represent diffusive states, where (D) is the diffusion coefficient. Immobile state (State 1), confined state (State 2) and highly mobile state (State 3). The areas of the circles represent the average state occupancy (%) of SVs in their respective states. The arrows indicate the transition probability of an SV moving from one state to the other. i Example of a trajectory from a recycling SV undergoing stochastic switching between the three diffusive states inferred by vbSPT analysis. Data in ( c – f ) are displayed as mean ± SEM. Values were obtained from n = 15 neurons (Recycling pool), and n = 16 neurons (Total pool), from over 3 independent neuronal cultures. 131 presynapses were analyzed in ( g ) and 35 presynapses were analyzed in ( h ). Statistical comparisons in ( d , f ) were performed using unpaired two-tailed Student’s t -test with Welch’s correction. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-MAP2 guinea pig polyclonal antibody (1:2500, Synaptic Systems, #188004), anti-VAMP2 mouse monoclonal antibody (1:500, Synaptic Systems, #104211), anti-Rab5 rabbit monoclonal antibody (1:1000, Abcam, #ab218624), anti-KDEL rabbit monoclonal antibody (1:200, Abcam, #ab176333), anti-Lamp1 mouse monoclonal antibody (1:100, Abcam, #ab25630), anti-GFP chicken polyclonal antibody (1:1000, MERK, #AB16901), anti-Tau mouse monoclonal antibody T46 (1:100, ThermoFisher, #13–6400), and anti-Tau clone Tau-5 mouse monoclonal antibody (1:1000, Sigma-Aldrich -Merck, #MABN162).

Techniques: Expressing, Incubation, Fluorescence, Diffusion-based Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles

doi: 10.1038/s41467-023-43130-4

Figure Lengend Snippet: a , b (i) Epifluorescence image of VAMP2-pHluorin expressed in WT neurons ( a ) or Tau KO neurons ( b ) incubated with At647N-GBP nanobodies. Insets (red outline) on a presynapses are shown at higher magnification in (ii). (iii) Intensity maps of recycling SVs. c , e Average MSDs of recycling SVs ( c ) or total SVs ( e ) from WT (black) or Tau KO (red) neurons. d , f Plots of the area under the MSD curves (AUC). g (i) Epifluorescence image VAMP2-pHluorin expressed in Tau KO neurons incubated with At647N-GBP nanobodies. Empty mEos2 was expressed as a control. Inset (red outline) on a presynapse is shown at higher magnification in (ii) (trajectory map of SVs). h (i) Epifluorescence image of VAMP2-pHluorin expressed in Tau KO neurons where Tau-mEos2 was re-expressed, and incubated with At647N-GBP nanobodies. Inset (red outline) on a presynapse is shown at higher magnification in (ii) (trajectory map of SVs). i Average MSD of recycling SVs from neurons expressing mEos2 (black) or Tau-mEos2 (red). j The corresponding area under the MSD curves (AUC). k Average MSD of recycling SVs from Tau WT neurons treated with control vehicle (Veh, DMSO; black), okadaic acid (1 μM, 15 min, OkAc; red), or staurosporine (1 μM, 15 min, Staur; blue). l The corresponding area under the MSD curves (AUC). m Average MSD of recycling SVs from Tau KO neurons treated with control vehicle (Veh, DMSO; black), okadaic acid (OkAc; red), or staurosporine (Staur; blue). n The corresponding area under the MSD curves (AUC). Data are displayed as mean ± SEM. Values were obtained from n = 13 and 16 neurons in ( c , d ), n = 17 and 18 neurons in ( e , f ), n = 21 and 31 neurons in ( i , j ), n = 16, 10 and 10 neurons in ( k , l ), and n = 11, 12 and 18 neurons in ( m , n ). Data was obtained from >2 independent neuronal cultures. Statistical comparisons were performed using the unpaired two-tailed Student’s t -test in ( d ) and ( f ) and the unpaired two-tailed Mann-Whitney U test in ( j ), and using the one-way ANOVA test followed by Dunnett post hoc test comparing the groups to the control (Veh) in ( l ) and ( n ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-MAP2 guinea pig polyclonal antibody (1:2500, Synaptic Systems, #188004), anti-VAMP2 mouse monoclonal antibody (1:500, Synaptic Systems, #104211), anti-Rab5 rabbit monoclonal antibody (1:1000, Abcam, #ab218624), anti-KDEL rabbit monoclonal antibody (1:200, Abcam, #ab176333), anti-Lamp1 mouse monoclonal antibody (1:100, Abcam, #ab25630), anti-GFP chicken polyclonal antibody (1:1000, MERK, #AB16901), anti-Tau mouse monoclonal antibody T46 (1:100, ThermoFisher, #13–6400), and anti-Tau clone Tau-5 mouse monoclonal antibody (1:1000, Sigma-Aldrich -Merck, #MABN162).

Techniques: Incubation, Control, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles

doi: 10.1038/s41467-023-43130-4

Figure Lengend Snippet: a , b Representative Tau-mEos2 trajectories generated using segNASTIC showing instantaneous diffusion coefficients (the color bar represents log 10 [μm 2 s -1 ]) in an ( a ) synaptic region and ( b ) an axonal region, determined by VAMP2-pHluorin intensity (inset), of neurons treated with control vehicle (DMSO) or with 1,6-HD (2.5%). c Average MSD of Tau-mEos2 presynaptic trajectories from neurons treated with control vehicle or with 1,6-HD. d Corresponding areas under the MSD curves (AUC). e Average MSD of Tau-mEos2 axonal trajectories from neurons treated with control vehicle or with 1,6-HD. f Corresponding areas under the MSD curves (AUC). g Representative segNASTIC trajectories showing instantaneous diffusion coefficients from Tau-mEos2, and Tau LLPS mutants Tau I277P / I308P -mEos2 and ΔTau74-mEos2. h , j Average MSD of the trajectories of mEos2-tagged Tau WT and LLPS Tau mutants in ( h ) presynapses and ( j ) axons. i , k Corresponding areas under the curve (AUC), in the ( i ) presynapses and ( k ) axonal compartment. Data in ( c ) to ( f ) and ( h ) to ( k ) are displayed as mean ± SEM. Data in ( c , d ) was obtained from n = 9 synapses from 9 different neurons. Data in ( e , f ) was obtained from n = 7 and 5 axons from different neurons. Data in ( h ) to ( k ) was obtained from n = 14 presynapses from 14 neurons and 18 axons from 18 neurons in Tau WT , n = 11 synapses and axons from 11 neurons in Tau I277P / I308P , and n = 17 synapses from 17 neurons and 12 axons from 12 neurons in ΔTau74. Data was obtained from 2 independent neuronal cultures. Statistical comparisons in ( d ) and ( f ) were performed using the unpaired two-tailed Student’s t -test with Welch’s correction in ( d ) and ( f ), or using the one-way ANOVA test in ( i ) and ( k ). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-MAP2 guinea pig polyclonal antibody (1:2500, Synaptic Systems, #188004), anti-VAMP2 mouse monoclonal antibody (1:500, Synaptic Systems, #104211), anti-Rab5 rabbit monoclonal antibody (1:1000, Abcam, #ab218624), anti-KDEL rabbit monoclonal antibody (1:200, Abcam, #ab176333), anti-Lamp1 mouse monoclonal antibody (1:100, Abcam, #ab25630), anti-GFP chicken polyclonal antibody (1:1000, MERK, #AB16901), anti-Tau mouse monoclonal antibody T46 (1:100, ThermoFisher, #13–6400), and anti-Tau clone Tau-5 mouse monoclonal antibody (1:1000, Sigma-Aldrich -Merck, #MABN162).

Techniques: Generated, Diffusion-based Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles

doi: 10.1038/s41467-023-43130-4

Figure Lengend Snippet: a (i) Representative epifluorescence image of a WT hippocampal neuronal segment expressing VAMP2-pHluorin and Tau-mEos2, used for imaging under resting conditions (Low K + ) and after stimulation (High K + ). (ii) The inset of the presynaptic compartment in resting condition is shown at a higher magnification. (iii) Trajectory map of Tau-mEos2 in the unstimulated presynaptic compartment. (iv) The inset of the presynaptic compartment during stimulation is shown at a higher magnification. (v) Trajectory map of Tau-mEos2 in the stimulated presynaptic compartment. b Plot showing the total number of Tau-mEos2 trajectories within the presynapse, in resting conditions (black) and after stimulation (red). c Plot showing the total number of Tau-mEos2 trajectories within the axon, in resting conditions (black) and after stimulation (red). d – g Average MSD of total Tau-mEos2 trajectories and the corresponding area under the curve (AUC) in resting conditions (black) and after stimulation (red), in the presynapse ( d , e ) and in the axonal compartment ( f , g ). h Trajectory map of (i) recycling SVs and (ii) Tau-mEos2 in the unstimulated presynaptic compartment, obtained by dual-color single-tracking super-resolution imaging. (iii) Merged image. i Trajectory map of (i) recycling SVs and (ii) Tau-mEos2 in the stimulated presynaptic compartment, obtained by dual-color single-tracking super-resolution imaging. (iii) Merged image. j , k Representative Tau-mEos2 trajectories generated using segNASTIC showing instantaneous diffusion coefficients (the color bar represents log 10 [μm 2 s -1 ]) in a presynaptic region, determined by VAMP2-pHluorin intensity (inset), of a neuron that was unstimulated ( j ) and subsequently stimulated ( k ). l Percentage of Tau-mEos2 trajectories that are clustered. m Average cluster density of Tau-mEos2. n Average MSD of clustered Tau-mEos2 trajectories represented as the area under the curves (AUC). o Average apparent lifetime of Tau-mEos2 clusters. Data are displayed as mean ± SEM. Values were obtained from n = 29 synapses in ( b , e ), n = 23 axons in ( c , g ), and n = 15 synapses in ( l , m , n , o ). Data was obtained from ≥2 independent neuronal cultures. Statistical comparisons were performed using the two-tailed Student’s paired t -test. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-MAP2 guinea pig polyclonal antibody (1:2500, Synaptic Systems, #188004), anti-VAMP2 mouse monoclonal antibody (1:500, Synaptic Systems, #104211), anti-Rab5 rabbit monoclonal antibody (1:1000, Abcam, #ab218624), anti-KDEL rabbit monoclonal antibody (1:200, Abcam, #ab176333), anti-Lamp1 mouse monoclonal antibody (1:100, Abcam, #ab25630), anti-GFP chicken polyclonal antibody (1:1000, MERK, #AB16901), anti-Tau mouse monoclonal antibody T46 (1:100, ThermoFisher, #13–6400), and anti-Tau clone Tau-5 mouse monoclonal antibody (1:1000, Sigma-Aldrich -Merck, #MABN162).

Techniques: Expressing, Imaging, Generated, Diffusion-based Assay, Two Tailed Test