Condensates of synaptic vesicles and synapsin are molecular beacons for actin sequestering and polymerization

Abstract

Neuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation (LLPS). This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. The reorganization of SVs, synapsins and actin is a hallmark of synaptic activity, but their interplay is still unclear. Here, we combined the reconstitution approaches, expansion microscopy, super-resolution imaging and cryo-electron tomography to dissect the roles of synapsin-SV condensates in the organization of the presynaptic actin cytoskeleton. Our data indicate that LLPS of synapsin initiates actin polymerization, allowing for SV:synapsin:actin assemblies to facilitate the mesoscale organization of SV clusters along axons mimicking the native presynaptic organization in both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-SVs condensates is an essential building block on a roadmap to unravel how coordinated neurotransmission along the axon enables circuit function and behavior.

Figure 1.. Synapsin 1 condensates are sites for actin polymerization.

A. Representative confocal images from the in-vitro reconstitution of EGFP-Syn1-FL (4 μM, 3% (w/v) PEG 8,000) with ATTO647 labelled G-actin monomers (4 μM) at t = 0 (left) and 35 min (right). Buffer used in these experiments, hereafter referred to as ‘reaction buffer’, contained: 25 mM Tris-HCl (pH - 7.4), 150 mM NaCl, 0.5 mM TCEP. Note the condensates growing over time and forming radial “asters.” Scale bar, 5 μm. B. Confocal images of the EGFP-Syn1-IDR (4 μM, 3% (w/v) PEG 8,000) and ATTO647 labelled G-actin reconstitution (4 μM) in reaction buffer at t = 0 (left) and 35 min (right). Scale bar, 5 μm. C. Plot for the quantification of normalized enrichment of actin within EGFP-Syn1-FL phases. Gompertz growth curve: Y = YM*(Y0/YM)^(exp(−K*X)), YM = 0.9611,Y0 = 0.01032 , K = 0.2958, 1/K = 3.381 , R² = 0.9376. Data shown here is quantified from 3 independent experiments and 30 analyzed condensates (N = 3, n = 30). Error bars represent SD. D. Plot showing the number of Sholl intersections for polymerized actin in the presence of EGFP-Syn1-FL, EGFP-Syn1-IDR and EGFP-Syn1-Dom. C condensates as a function of distance (in μm). The number of Sholl intersections was counted in 0.5 μm radius increments from the center of the condensates. Each dot represents the mean intersections count for each condition. Error bars represent SEM. E. Plot depicting the quantification of pyrene-actin fluorescence intensity as a function of time. Pyrene-actin (10 μM) polymerization was assessed in the presence of EGFP-Syn1-FL or EGFP-Syn1-IDR (10 μM) in 1X actin polymerization buffer (10 mM Tris/HCl, pH = 7.5, 2 mM MgCl₂, 50 mM KCl and 0.5 mM ATP). The data shown here is quantified from 3 independent experiments (N = 3). Error bars represent SD.

Figure 2.. Actin torus formation around Syn1-FL condensates precedes aster assembly.

A. Scheme and corresponding representative confocal images from the in vitro Syn1-actin reconstitution assay showing actin enrichment over a period of 20 min within EGFP-Syn1-FL condensates. Images were acquired at excitation wavelengths 488 nm and 647 nm for EGFP-Synapsin 1-FL and ATTO647 G-actin respectively. Scale bar, 5 μm. B. Scheme and corresponding representative confocal images depicting stages of “actin-aster” assembly from EGFP-Syn1-FL condensates. Scale bar, 5 μm. Here, the same datasets are shown with two different LUTs to highlight ‘torus’ and ‘aster’ structures. Right: line profiles depict the enrichment of fluorescently labelled actin within the condensate. Data obtained from 3 independent reconstitutions, 30 condensates analyzed for each stage. Error bars represent SEM. C. Scheme of “actin-asters” connecting and forming “web-like” assembly and corresponding confocal image of polymerized “actin-asters” forming “web-like” structures but with different LUTs to highlight ‘torus’ and ‘aster’ structures. Scale bar, 10 μm. D. Representative images from the EGFP-Syn1-FL and actin reconstitution acquired using TIRF microscopy show “actin-toruses” and “actin-fibers” as indicated with orange and blue arrows respectively. Scale bar, 5 μm.

Figure 3.. The morphology of synapsin 1-actin assemblies as a function of synapsin 1 phosphorylation.

A. 3D synapsin 1 protein structure prediction, obtained from the AlphaFold protein structure database, entry AF-O88935-F1-model_v4. Arrows point the serine residues that are targeted by PKA (S9) and CaMKII (S568 and S605) in mouse synapsin 1. For the reconstituted protein variants used in this study, Syn 1-PKA has serine 9 mutated to glutamate, Syn 1-CaMKII has both serines 568 and 605 mutated, and EGFP-Syn 1-PKA/CaMKII has all three serines mutated. B. Representative images for actin polymerization reactions in the presence of purified EGFP-Syn 1, EGFP-Syn 1-PKA, EGFP-Syn 1-CaMKII and EGFP-Syn 1-PKA/CaMKII, with 3% PEG 8K in reaction buffer. All images were taken after 40 min of incubation. Images were acquired using spinning-disk confocal microscope, employing the 647 nm wavelength for actin (represented in green). Scale bars, 10 μm. C. Graph showing the number of Sholl intersections for polymerized actin in the presence of EGFP-Syn1, EGFP-Syn1-PKA, EGFP-Syn1-CaMKII and EGFP-Syn1-PKA/CaMKII condensates as a function of distance (in μm). The number of Sholl intersections was counted in 0.5 μm radius increments from the center of the condensates, with at least 100 synapsin:actin assemblies analyzed for each condition. Each dot represents the mean intersections count for each condition. Data from 3 independent reconstitutions analyzed. Error bars represent the SEM. For statistical analysis, an R code for linear mixed effect model was applied, as in (Jackson et al, 2024); The code is available at (https://zenodo.org/records/1158612) (Wilson et al, 2017). ***: p<0.001, **: p<0.01, *: p<0.05.

Figure 4:. Actin forms toruses at the interface of SV condensates in vitro and in living synapses.

A. Left: Representative images of the reconstituted EGFP-Syn1-FL (4 μM, 3% (w/v) PEG 8,000) and SVs (3 nM, labelled with FM4-64, 1.65 μM) condensates after adding ATTO647 labelled G-actin monomers (4 μM) at t = 0 (top) and 35 min (bottom). Images were acquired using a spinning disk confocal microscope. Right: Magnified images of polymerized actin-asters formed at 35 min. Scale bar, 5 μm. B. Scheme of SV recruitment along the actin fibers. C. Cartoon illustrating the lamprey spinal cord with giant reticulospinal (RS) axons and microinjection strategy. V, ventral; D, dorsal. D. Laser-scanning confocal microscopy (LSM) image of a live lamprey reticulospinal axon co-injected with FM4-64 and Alexa Fluor^™ 488-phalloidin for characterizing SVs and actin, respectively. Excitation wavelengths were 488 nm for phalloidin-actin and 560 nm for SVs labelled with FM4-64. The Inset shows the clear assembly of actin as a torus around SVs. Scale bars, 5 μm (overview) and 1 μm (insets). E. LSM images of immunolabeled lamprey synapses. Synapses were stained for endogenous synapsin 1, endogenous SV2 (an SV marker), and actin (Alexa Fluor^™ 488-phalloidin). Scale bar, 1 μm. F. High resolution confocal microscopy maximum z-projection image showing individual synapses connected together by phalloidin labeled “aster-like” structures (white arrows). Spinal cord was bathed in ringer solution containing 0.03% DMSO followed by injection of Alexa Fluor^™ 488-phalloidin. The same image is depicted twice using the indicated minimum and maximum values (16-bit scale). Scale bar, 2 μm.

Figure 5:. The effects of the inhibition of actin polymerization on presynaptic actin assemblies in-vitro and in living synapses.

A. Schematic illustrating the experimental outline for the reconstitution of G-actin monomers pre-treated with Latrunculin A (Lat A) with the subsequent addition of synapsin 1and 3% PEG 8,000. B. Representative maximum intensity projection from confocal microscopy images from the reconstitution of 4 μM Lat A treated G-actin monomers with synapsin 1 condensates in reaction buffer at t = 0, 2, 15 and 30 min. Images were acquired at 488 and 647 nm wavelengths for EGFP-synapsin 1 and ATTO647 actin, respectively. Scale bar, 3 μm. C. Schematic illustrating the experimental outline for the addition of 4 μM Lat A on top of pre-formed “actin-asters” formed from synapsin 1 condensates. D. Representative maximum intensity projection from confocal microscopy images from the treatment of reconstituted “actin-asters” with 4 μM Lat A. Images were acquired both before and after (t = 0, 15 and 30 min) addition of 4 μM Lat A at 488 and 647 nm wavelengths for EGFP-synapsin 1 and ATTO647 actin, respectively. Scale bar, 3 μm. E. Schematic illustrating the experimental outline for the Lat A based experiments in lamprey reticulospinal (RS) axons ex-vivo. F. Treatment of lamprey RS synapses with Lat A before the injection of fluorescently conjugated phalloidin (Alexa Fluor 488-Phalloidin) leads to the disappearance of the presynaptic actin assemblies. Top: Representative maximum intensity z-projection image of presynaptic actin assemblies inside lamprey RS axons pre-treated with 0.03% DMSO (Control), followed by microinjection with Alexa Fluor 488-Phalloidin. Bottom: Representative maximum intensity z-projection image showing the absence of pre-synaptic actin assemblies inside lamprey RS axons pre-treated with 2.5 μM Lat A in 0.03% DMSO, followed by microinjection with Alexa Fluor 488-Phalloidin. Magnified regions (right) are illustrated in the overview section (left). White arrows point to aster formations. Scales bars = 10 μm (left) and 1 μm (right). G. Treatment of lamprey RS synapses with Lat A after the injection of Alexa Fluor 488-Phalloidin does not affect pre-synaptic actin assemblies. Top: Representative maximum intensity z-projection image of pre-synaptic actin assemblies inside lamprey RS axons microinjected with Alexa Fluor 488-Phalloidin and post-treated with 0.03% DMSO (Control). Bottom: Representative maximum intensity z-projection image of pre-synaptic actin assemblies inside lamprey RS axons microinjected with Alexa Fluor 488-Phalloidin and post-treated with 2.5 μM Lat A in 0.03% DMSO. Magnified regions (right) are illustrated in the overview sections (left). White arrows point to aster formations. Scales bars = 10 μm (left) and 1 μm (right). H. Bar chart showing the quantification of the density (number of objects per surface area of axon) of phalloidin-positive objects inside axons pre-treated with Lat A (2.5 μM in 0.03% DMSO) or the corresponding control (0.03% DMSO). Each dot represents a z-stack image series through an axon. Error bars represent standard error of the mean (SEM). Data is from n = 14 images; n = 3 axons from N = 3 animals. For statistical analysis, an unpaired t-test was performed in GraphPad PRISM. **** indicates a p-value <0.0001. I. Bar chart showing the quantification of the density of phalloidin-positive objects inside axons post-treated with Latrunculin A (2.5 μM in 0.03% DMSO) or the corresponding control (0.03% DMSO). Each dot represents a z-stack image series through an axon. Error bars represent standard error of the mean (SEM). Data is from n = 16-18 images; n = 3-4 axons from N = 3-4 animals. For statistical analysis, an unpaired t-test was performed in GraphPad PRISM. P-value = 0.5334.

Figure 6.. Actin aster are stable structures connecting neighboring condensates.

A. Representative confocal image from the reconstituted EGFP-synapsin 1:actin:GUV assemblies. Projection of the maximal intensity, 30 min post-incubation. Scale bar, 10 μm. B. Magnified region from A indicating the long actin fibers connecting the adjacent condensates. C. Top: Skeletonized actin fibrils from A. Bottom: A single continuous network of synapsin condensates interconnected with a continuous actin network is highlighted in green. Scale bar, 20 μm. D. Heatmap of co-dependencies (Pearson’s correlation) for synapsin1:actin assemblies' node properties: (1) volume, (2) surface area, (3) diameter, (4) main-, (5) second-, (6) third- axis length, (7) mean-, (8) maximum, (9) minimum- intensity, (10) median Euclidean distance from other nodes). Data obtained from 3 independent reconstitutions. E. Principal component (PC) analysis of synapsin1:actin assemblies: scatter plot of PC1 vs. PC2 scores (3 clusters identified by k-means clustering highlighted in different colors). Data obtained from 3 independent reconstitutions. F. Representative confocal image from the reconstituted synapsin 1:actin assemblies spreading over the surface of a GUVs. Projection of the maximal intensity, 30 min post-incubation. Scale bar, 3 μm. G. Schematic representation of the synapsin:actin:GUV association. H. Representative confocal images of synapsin:actin assemblies before (top) and after (bottom) the addition of 1,6-hexanediol (10% w/v). The treatment of the synapsin:actin assemblies with 1,6-hexanediol partially disperses synapsins without affecting the pre-formed actin asters. Asterisks indicate synapsin condensates without actin, which do disperse upon the addition of 1,6-hexanediol, as expected. Scale bars: 10 μm for the large images, 1 μm for the crop. I. Quantification of the synapsin 1 and actin partitioning before and after treatment with 1,6-hexanediol. Data from three independent reconstitutions, >1500 condensates analyzed for each condition. ****p<0.0001, ns not significant; Mann-Whitney nonparametric test.

Figure 7.. Actin accumulates at pre- and post- synapses in murine neurons.

A. Cryo-EM tomogram of a wild-type synapse. Scale bar, 100 nm. B. 3D rendering of structures identified in A. Annotation: yellow, synaptic vesicles (SVs); green, pre-synaptic plasma membrane; blue, post-synaptic plasma membrane; red, actin filament; colored arrow-heads point to actin filaments at the pre-synapse. C, D. exemplary region highlighting actin filaments associated with SV cluster from A. Note the reconstructed actin filaments (red in C) and original signal (arrowheads in D) juxtaposed to SVs. Scale bar, 50 nm. E. exemplary region highlighting actin filaments in the periactive zone associated with the plasma membrane. Scale bar, 50 nm. F. Expansion microscopy images of wild-type hippocampal neurons in culture (14 DIV) immunostained for VGLUT1 (an integral SV protein), PSD95 (post-synaptic protein), and actin. The inset (white dotted line) focuses on resolved pre- and post- synaptic regions. Note the presence of actin in both compartments (yellow line outlines the pre-synapse). Expansion factor 4.3; scale bar, 1 μm G. The same as in F but for neurons from synapsin triple knock-out animals (SynTKO). H. Quantification of the actin signal present in VGLUT1 positive regions in synapses from WT and SynTKO neurons. Data from three independent neuronal preparations, 731/1148 synapses analyzed for WT/TKO respectively. Bars represent mean ±SD; **** indicates p <0.0001. I. Quantification of the actin signal present in PSD95 positive regions in synapses from WT and SynTKO neurons. Data from three independent neuronal preparations, 1036/1215 synapses analyzed for WT/TKO respectively. Bars represent mean ±SD; n.s., non-significant.

Figure 8:. Synapsin-SV condensates are necessary to sequester actin in living synapses.

A,B. Two-color super-resolution STED images of hippocampal neurons (14 DIV) from A wild-type and B synapsin triple knockout neurons stained with StarRed-phalloidin and anti-synaptophysin (Star580). Scale bars, 1 μm. C. Magnified region from (A) showing a cohort of synaptic vesicles (anti-synaptophysin) colocalizing with actin (StarRed-phalloidin). Scale bar, 500 nm. D. Magnified region from (B) indicating a more dispersed signal of SVs (anti-synaptophysin) lacking the colocalization with actin (StarRed-phalloidin). Scale bar, 500 nm. E,F. Representative images of synapsin triple knockout neurons rescued with (E) full-length EGFP-synapsin 1 or (F) EGFP-synapsin 1 IDR and stained with StarRed-phalloidin and anti-synaptophysin (Star580). Scale bars, 1 μm. G,H. Magnified regions from (E) and (F), respectively, showing colocalization of SVs with actin. Scale bars, 500 nm. I. Autocorrelation of the actin signal along the axons (StarRed-phalloidin; see lines in the images in A and B) indicates that actin rings remain unaltered in the absence of synapsins. J. Actin enrichment in synaptic vesicle cohorts defined as a normalized intensity signal of actin channel within regions positive for synaptophysin (wild-type and synapsin triple knockout) or both synaptophysin and EGFP (in rescue experiments). Data from three independent neuronal preparations; data points present median maximum actin intensity per image. Mann-Whitney Test; WT-SynTKO; SynTKO-SynTKO+FL; SynTKO-SynTKO+IDR are all p<0.0001 (****).