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. 2021 Sep 8;14(1):137.
doi: 10.1186/s13041-021-00846-y.

Multivalent electrostatic pi-cation interaction between synaptophysin and synapsin is responsible for the coacervation

Goeun Kim  1 , Sang-Eun Lee  1   2 , Seonyoung Jeong  1 , Jeongkun Lee  1 , Daehun Park  3 , Sunghoe Chang  4
Affiliations

Multivalent electrostatic pi-cation interaction between synaptophysin and synapsin is responsible for the coacervation

Goeun Kim et al. Mol Brain. .

Abstract

We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid-liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi-cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph's negative charge, the results indicate that pi-cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi-cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

Keywords: Liquid–liquid phase separation (LLPS); Pi–cation interactions; Presynaptic nerve terminals; Synapsin; Synaptic vesicle cluster; Synaptophysin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Syph Ct contains repeated regions and alone forms liquid droplets when incubated at high concentrations in vitro. a Domain structure of full-length mouse Syph. Nt N-terminus, TM transmembrane domain, Ct C-terminus (amino acids 219–308). b Repeated sequence in the cytoplasmic domain synaptophysin Ct. Syph Ct contains 10 repeated regions, 9 of which start with tyrosine. The repeated sequences are aligned to show the consensus sequence Y-G-P/Q-Q-G. c The pie graph shows the proportion of Tyr, Gly, Pro and Gln residue in the Syph Ct. d The prediction plot of intrinsically disordered regions in the full-length Syph using PrDOS. The shaded region is Syph Ct, which is likely to be an IDR. e Fluorescence images showing droplet formation of purified Syph Ct-mCh alone (50 μM) in vitro in the presence of 10% PEG-8000 at RT. f Representative fluorescence image of Syph Ct-mCh expressed in COS-7 cells. Scale bars, 20 μm
Fig. 2
Fig. 2
Syph Ct undergoes phase separation among themselves when assisted by additional interactions in living cells. a Schematic diagram of Syph Ct-mCh-CRY2PHR consisting of the N-terminal Syph Ct (blue-gray) fused to mCherry (red) and the CRY2PHR domain (gray indicating inactive state). Blue light activation of Syph Ct-mCh-CRY2PHR leads to rapid clustering (blue indicating active CRY2PHR). b Representative time-lapse fluorescence images of light-activated clustering of Syph Ct-mCh-CRY2PHR and CRY2PHR-mCh stimulated with a 488 nm laser for 2500 ms. Middle: Magnified images of the region enclosed by a red rectangle in the top panel. Scale bars; 20 μm (top and bottom), 2 μm (middle). c Schematic diagram of Syph (Ct)2-mCer-MP. Two Syph Cts were linked by a short linker (gray) and fused to mCerulean fluorescent protein and the multimeric protein (MP) of CaMKIIα (pale mint). 12 identical MP subunits are assembled into a circular oligomer, exposing 24 copies of Syph Cts. d Representative fluorescence image of droplets formed by Syph (Ct)2-mCer-MP expressed in living cells. e Representative fluorescence image of droplets formed by purified Syph (Ct)2-mCer-MP (5 μM) in vitro in the presence of 3% PEG-8000. Scale bars; d = 20 μm, e = 10 μm. f Time-lapse images showed fusion of two Syph (Ct)2-mCer-MP droplets in living cells. g Representative fluorescence images of Syph (Ct)2-mCer-MP droplets treated with 3% 1,6-Hexanediol (3% 1,6-HD). Droplets disperse reversibly upon 3% 1,6-HD. Scale bars; f = 2 μm, g = 20 μm. h Representative time-lapse images showing fluorescence recovery of Syph (Ct)2-mCer-MP droplet after photobleaching. i Plot of the average fluorescence intensities after photobleaching of multiple spots. N = 10 cells from 5 coverslips. Scale bars; 2 μm
Fig. 3
Fig. 3
Phase separation of Syph Ct alone is driven by tyrosine-tyrosine interactions. a Schematic diagram of Syph Ct WT and 9YS mutant. Y245, Y250, Y257, Y263, Y269, Y273, Y284, Y290, and Y295 were mutated to serine (9YS). b Representative time-lapse fluorescence images of light-activation of Syph Ct 9YS-mCh-CRY2PHR in COS-7 cells. c, d Representative fluorescence image of Syph (Ct)2 9YS-mCer-MP expressed in COS-7 cells (c) and purified Syph (Ct)2 9YS-mCer-MP at 10 μM in vitro in the presence of 3% PEG-8000 (d). Scale bars, 20 μm
Fig. 4
Fig. 4
pi–cation electrostatic interactions govern the coacervating behavior between Syph and positively charged proteins. a Representative fluorescence images of co-condensates formed by purified Syph Ct-mCh and FUS-RBD-mEGFP in vitro. Syph Ct-mCh (5 μM) and FUS-RBD-mEGFP (2.5 μM) were mixed with 5% PEG-8000. b COS-7 cells were transfected with Syph-HA and mCh-Syn (top) or Syph 9YS-HA and mCh-Syn (bottom), and Syph was detected by immunostaining of HA (ICC). Unlike Syph-HA, no droplets were observed with Syph 9YS expression. Scale bars, 20 μm
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