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Assembly of synaptic active zones requires phase separation of scaffold molecules

Nature volume 588, pages 454–458 (2020)Cite this article

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Abstract

The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system1. Every presynapse builds a core ‘active zone’ structure, where ion channels cluster and synaptic vesicles release their neurotransmitters2. Although the composition of active zones is well characterized2,3, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-α) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.

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Fig. 1: The active-zone scaffold proteins SYD-2 and ELKS-1 are dynamic at nascent synapses and capable of phase separation.
Fig. 2: Identification and mutation of the phase-separation motifs of SYD-2 and ELKS-1.
Fig. 3: Phase separation of SYD-2 and ELKS-1 is required for assembly of the presynaptic active zone.
Fig. 4: The fluidity of SYD-2 and ELKS-1 condensates facilitates the incorporation of active-zone components.

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Acknowledgements

We thank L. Luo, S. Chen and members of the Shen laboratory for critical reading of the manuscript; J. Lee for the GFP–SYD-2 strain; K. M. Collins for HSN strains; P. Calsou and N. L. Fawzi for FUS constructs; E. M. Jorgensen and B. Goldstein for FLPon tools; M. Boxem for a tagBFP2 construct; and L. Feng and Y. Xu for assistance with size-exclusion chromatography. This work was funded by Howard Hughes Medical Institute, of which K.S. is an investigator; by NIH grant R37 NS048392 to K.S.; and by a Helen Hay Whitney postdoctoral fellowship to N.A.M.

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Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, CA, USA

    Nathan A. McDonald & Kang Shen

  2. Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA, USA

    Richard D. Fetter & Kang Shen

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  1. Nathan A. McDonald
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Contributions

N.A.M. and K.S. conceptualized the study. N.A.M. carried out the experiments and analyses. R.D.F. carried out electron-microscopy experiments and analyses. N.A.M. wrote the paper. N.A.M., R.D.F. and K.S. edited the paper.

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Correspondence to Kang Shen.

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Peer review information Nature thanks Erik Jorgensen, Dragomir Milovanovic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Presynaptic active-zone scaffolds display LLPS characteristics.

a, Predictions of intrinsic disorder in the sequences of presynaptic active-zone components, using the IUPred2a (ref. 37) and Spot-Disorder2 (ref. 38) algorithms. Values above 0.5 indicate intrinsic disorder. b, Predictions of LLPS of active-zone components, using catGRANULE39 and Pi–Pi predictor25 algorithms. Higher values indicate a propensity towards phase separation. c, Expression of GFP- or mCherry-tagged presynaptic active-zone components in COS-7 cells. FUS–GFP is a positive control40,41. Red boxes indicate example puncta that are photobleached in d. Scale bar, 10 μm. d, FRAP analysis of puncta. Data are means ± 95% CI from two independent experiments. Insets show example recovery kinetics of puncta from c. Scale bar, 1 μm. e, Time-lapse analysis of in vivo droplets containing SYD-2 or ELKS-1, showing liquid behaviours of fusion and relaxation. Red arrowheads point to droplets before fusion. Liquid behaviour was observed in multiple cells and in different technical replicates. UNC-10 puncta were dynamic when analysed by FRAP, but never showed liquid behaviour. Scale bars, 1 μm.

Source data

Extended Data Fig. 2 Controls for in vitro phase separation assays.

a, In vitro LLPS assay workflow and scoring conditions. b, Size-exclusion chromatography of purified mCherry and mCherry–FUS in 20 mM Tris pH 7.4, 500 mM NaCl, indicating that the protein isolated by the high-salt purification method is soluble. A280, absorption at 280 nm. c, In vitro LLPS assay. Left panels, soluble input materials before dilution to physiological salt concentration. Right panels, comparison between the effects of no crowding and 10% PEG conditions on a known phase-separating motif (FUS)41 and a negative control (mCherry). d, Representative FRAP images (top) and quantified FRAP dynamics (bottom) of FUS and mCherry in vitro condensates formed in 10% PEG. Data are means ± 95% CI from three independent experiments. e, Time-lapse analysis of in vitro condensates containing mCherry and FUS, showing the presence or absence of liquid behaviour. Phase-separation assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl and 10% PEG 3350 where indicated with 10 μM protein. f, g, Phase diagrams for mCh–SYD-2(Nter) (f) and mCh–ELKS-1 (g), showing protein concentrations versus NaCl or PEG concentrations. Each combination was tested three times. Scale bars, 5 μm.

Source data

Extended Data Fig. 3 Purified constructs for in vitro phase separation assays.

SDS–PAGE gels, stained by Coomassie blue, of the purified constructs used for in vitro assays in the indicated figures. Many constructs display a secondary band 10 kDa below the major species, due to mCherry cleavage at the fluorophore centre (Y72)33.

Extended Data Fig. 4 Structure–function analysis of SYD-2 phase separation.

a, Diagram showing the structure of SYD-2, including disordered regions and coiled-coils predicted by the indicated algorithms37,38,42. LH1/LH2, liprin-homology domains; SAM, sterile alpha motif domains. b, Purified recombinant mCherry–SYD-2 constructs tested in an in vitro phase-separation assay. mCherry was fused to the N terminus of each sequence. c, Left panels in each column, example time-lapse analyses of in vitro condensates comprising the indicated SYD-2 constructs, showing the presence or absence of liquid fusion behaviour. Right panels, FRAP dynamics of each SYD-2 condensate. Data are means ± 95% CI for at least three condensates from two independent experiments. The y-axes show percentage intensity. Phase-separation assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl with 10 μM protein. FRAP assays were performed on condensates formed in crowding conditions of 10% PEG, except in the case of the blue curves, where there was no crowding agent. Green or blue curves indicate a positive result (scored LLPS+) and black curves a negative result (scored LLPS−). Scale bars, 5 μm.

Source data

Extended Data Fig. 5 Structure–function analysis of ELKS-1 phase separation.

a, Diagram showing the structure of ELKS-1, including disordered regions and coiled-coils predicted by the indicated algorithms37,38,42. b, Purified recombinant mCherry–ELKS-1 constructs tested in an in vitro phase-separation assay. mCherry was fused to the N terminus of each sequence. c, Left panels in each column, example time-lapse analyses of in vitro condensates comprising the indicated ELKS-1 constructs, showing the presence or absence of liquid fusion behaviour. Right panels, FRAP dynamics for each ELKS-1 condensate. Data are means ± 95% CI, for at least three condensates from independent experiments. The y-axes show percentage intensity. Phase-separation assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl with 10 μM protein. FRAP assays were performed on condensates formed in crowding conditions of 10% PEG, except in the case of the blue curves, where there was no crowding agent. Green or blue curves indicate a positive result (scored LLPS+) and black curves a negative result (scored LLPS−). Scale bars, 5 μm.

Source data

Extended Data Fig. 6 Phase-separation mutants of SYD-2 and ELKS-1 retain the ability to bind partners.

Shown are the results of yeast two-hybrid assays using the indicated bait and prey constructs. Protein–protein interactions are indicated by growth on −His/−Ade media.

Extended Data Fig. 7 Phase separation of SYD-2 and ELKS-1 is required for the assembly of multiple presynaptic active-zone components.

a–c, Cell-specific endogenous markers of presynaptic active zones in GFP–SYD-2 (a), GFP–ELKS-1 (b) or GFP–SYD-2(IDR∆) plus elks-1(LLPS–) (c) worms. d, Cell-specific endogenous markers of presynaptic active zones in syd-2 and elks-1 complete loss-of-function mutants. e, Quantified intensity of endogenous active-zone markers, as well as GFP–SYD-2 and GFP–ELKS, at HSN synapses in the indicated genotypes. Data are means ± 95% CI from independent animals. Comparisons were made using one-way ANOVA and Dunnett’s test. f, Expression of SNB-1 labelled with yellow fluorescent protein (YFP) from the unc-86 promoter at HSN synapses and cell bodies43. g, Quantification of SNB-1–YFP at HSN synapses and cell bodies. Data are means ± 95% CI from independent animals. Comparisons were made using one-way ANOVA and Dunnett’s test. h, Localization of endogenous GFP–SYD-2 or GFP–SYD-2(IDR∆) in the indicated genetic backgrounds. i, Quantification of endogenous GFP–SYD-2 or GFP–SYD-2(IDR∆) at HSN synapses in the indicated genetic backgrounds. Data are means ± 95% CI from independent animals; comparisons were made using one-way ANOVA and Dunnett’s (versus wild type) or Tukey’s (within genotypes) tests. Data for GFP–SYD-2, GFP–SYD-2(IDR∆) and GFP–SYD-2; elks-1(tm1233) are from Extended Data Fig. 7e. Dotted outlines show synaptic regions. Scale bars, 5 μm.

Source data

Extended Data Fig. 8 Phase separation of SYD-2 and ELKS-1 is required for proper synaptic function.

a, Left, the C. elegans egg-laying neuronal circuit44. The hermaphrodite-specific neuron (HSN) extends an axon across the vulval region, making specific synaptic contacts with vm1/2 vulval muscles and inhibitory VC4/5 motor neurons. Right, experimental setup. HSN is activated by channelrhodopsin ChR2 with blue light, while contraction of the vm2 muscle is measured with the Ca2+ sensor GCaMP5 (ref. 21). b, Representative vulval-muscle Ca2+ traces, detected by GCaMP5, in the indicated genotypes. ‘−ATR’ conditions lacked the essential ChR2 cofactor all-trans retinol. c, Quantification of the Ca2+ spike frequency, measured using GCaMP5, in the indicated genotypes. Data are means ± 95% CI from independent animals, with comparisons made using one-way ANOVA and Dunnett’s test.

Source data

Extended Data Fig. 9 Presynaptic assembly phenotypes of syd-2(IDR∆) mutants are rescued by certain exogenous phase-separation motifs only.

a, Diagram showing the replacement constructs tested. b, Comparison of amino-acid complexity between SYD-2/ELKS-1 and FUS/hnRNPA2 LLPS motifs. c, Left, time-lapse analysis of in vitro condensates comprising rescue constructs, showing liquid behaviour. Right, FRAP dynamics of in vitro condensates comprising rescue constructs. Data are means ± 95% CI across two independent experiments. SAM domains were omitted in these constructs. Assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl with 10 μM protein. d, e, Cell-specific endogenous markers of presynaptic active zones (UNC-10, d) or synaptic vesicles (RAB-3, e) in the indicated GFP–SYD-2 alleles. Dotted boxes show synaptic regions. f, Quantified intensity of UNC-10 and RAB-3 at HSN synapses in the indicated genotypes. Data are means ± 95% CI from independent animals, with comparisons made using one-way ANOVA and Dunnett’s (versus wild type) or Tukey’s (IDR∆ versus IDR∆ + FUS) tests. Data for wild type, syd-2(IDR∆) and syd-2(IDR∆ + FUS) are from Fig. 3. g, Quantification of egg stages laid by animals of the indicated genotypes. The syd-2(IDR∆ + hnRNPA2 construct rescues egg laying only in the presence of mScarlet-I–FLPon–RAB-3, for unknown reasons. Data are means ± 95% CI from independent animals. Data for wild type, syd-2(IDR∆) and syd-2(IDR∆ + FUS) are from Fig. 3. Scale bars, 5 μm.

Source data

Extended Data Fig. 10 Loss of liquidity of SYD-2 and ELKS-1 impairs the scaffolding of GIT-1.

a, Endogenous GFP–ELKS-1(LLPS–) at early (1.5-fold embryo) nerve-ring synapses. Insets show FRAP images of bleached synapses. Main scale bar, 5 μm; inset scale bars, 1 μm. b, Quantification of nerve-ring GFP–ELKS-1 FRAP dynamics. Data are means ± 95% CI across two independent experiments. Wild-type data are from Fig. 1. c, FRAP dynamics of in vitro condensates comprising SYD-2 and ELKS-1 fragments. Data are means ± 95% CI from two independent experiments. Phase-separation assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl and 10% PEG 3350, with 10 μM protein. Data for the ‘<5 min’ curve are from Fig. 2. d, Diagram showing the in vitro scaffolding experiment. e, f, Left, fluorescence images of the indicated condensates. Right, linescans performed through the centre of each condensate. Scale bars, 5 μm. g, Incorporation index (Iinc) for GIT-1–Dylight633. Data are means ± 95% CI from three independent experiments, with comparisons made using one-way ANOVA and Dunnett’s test.

Extended Data Fig. 11 Timing of assembly of the presynaptic active zone in early nerve-ring synapses.

Localization of endogenous GFP-tagged presynaptic active-zone components at sequential stages of embryo development. Each panel shows a maximum z projection of a separate embryo. Embryos are outlined in blue. A zy resliced projection through the nerve-ring region is shown to the right of each image. Red asterisks and arrowheads indicate visible localization to the nerve ring. Most puncta visible throughout the embryo are background autofluorescence, present even in untagged embryos (bottom). The timing of the emergence of fluorescence is stereotypical and was replicated in a second experiment. Scale bars, 10 μm.

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Supplementary Table 1. C. elegans strains used in this study.

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Supplementary Table 2. Plasmids used in this study.

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McDonald, N.A., Fetter, R.D. & Shen, K. Assembly of synaptic active zones requires phase separation of scaffold molecules. Nature 588, 454–458 (2020). https://doi.org/10.1038/s41586-020-2942-0

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