The Liprin homology domain is essential for the homomeric interaction of SYD-2/Liprin-α protein in presynaptic assembly

Abstract

Synapses are asymmetric structures that are specialized for neuronal signal transduction. A unique set of proteins is present at the presynaptic active zone, which is a core structure essential for neurotransmitter release. In Caenorhabditis elegans HSN neurons, SYD-2, a Liprin-α family protein, acts together with a GAP protein SYD-1 to promote presynaptic assembly. Previous studies have shown that elevating the activity of syd-2 can bypass the requirement of syd-1. Liprin-α proteins are composed of coiled-coil-rich regions in the N-terminal half, which mediate interactions with adapter proteins at the presynaptic active zone, and three SAM domains in the C terminus, which bind proteins such as LAR receptor tyrosine phosphatase. To address the molecular mechanism by which SYD-2 activity is regulated, we performed structure-function studies. By monitoring the ability of SYD-2 transgenes to rescue syd-2(lf) and to suppress syd-1(lf) phenotypes in HSN neuron synapses, we identified the N-terminal half of SYD-2 as minimally required for rescuing syd-2(lf) phenotypes. A highly conserved short coiled-coil segment named Liprin Homology 1 (LH1) domain is both necessary and sufficient to suppress syd-1(lf) defects. We show that the LH1 domain forms a dimer and promotes further oligomerization and/or complex formation of SYD-2/Liprin-α proteins. The role of the LH1 domain in presynaptic assembly can be partially complemented by artificial dimerization. These findings suggest a model by which the self-assembly of SYD-2/Liprin-α proteins mediated by the coiled-coil LH1 domain is one of the key steps to the accumulation of presynaptic components at nascent synaptic junctions.

Figure 1.

SYD-2/Liprin-α proteins and summary of transgenic analyses. A, Illustration of the domain structure of C. elegans SYD-2 and human Liprin-α1 proteins. Homology (percentage of identical residues) and predicted coiled-coil segments (underline) are indicated. B, Sequence alignment of the LH1 domains of SYD-2 and human Liprin-α proteins. The residues with black and gray background represent identical and similar residues, respectively. Asterisk indicates Arg184 residue of SYD-2. C, Summary of transgenic structural-functional analyses of SYD-2. The structure of SYD-2/Liprin-α constructs and the presence (+) or absence (−) of the ability to rescue syd-2(lf) or suppress syd-1(lf) phenotypes are indicated. Transgenic lines and plasmids used for pan-neural and HSN expression (#) are listed.

Figure 2.

The conserved coiled-coil LH1 domain is necessary for SYD-2 function. A, Illustration of the HSN neuron and synapses. The neurite (bold gray line) normally forms synapses at vulva, which can be visualized as clusters of fluorescent synaptic markers (black dots). The dotted rectangle corresponds to the area shown in the images. B–F, Activity of pan-neurally expressed SYD-2 transgenic constructs to rescue syd-2(lf) phenotype. B, Localization of synaptic vesicle markers SNB-1::YFP and GFP::RAB-3, presynaptic active zone markers GIT-1::YFP and ELKS-1::GFP at vulval synaptic region in HSN in wild-type, syd-2(ju37) and syd-2(ju37) with SYD-2 transgenes. C, Quantification of SNB-1::YFP localization at HSN synapses. D, Quantification of egg-laying behavior. Values represent percentage of animals (mean ± SD of independent lines, and n = the numbers of animals scored). E, F, Quantification of fluorescence intensity of GFP::RAB-3 (E) and GIT-1::YFP (F) puncta at synaptic regions. Values represent relative intensity (mean ± SEM, 5 animals). G–I, Expression of mCherry-tagged SYD-2 transgenic constructs in HSN. G, Localization of mCherry::SYD-2 constructs (full-length, N719, LH1, and ΔLH1) at HSN synaptic region in wild type and syd-2(ju37). H, Quantification of mCherry::SYD-2 localization. I, Quantification of egg-laying behavior. Values represent percentage of animals (mean ± SD of independent lines, n = the numbers of animals scored). **p < 0.01, *p < 0.05 [Fisher's exact test with Holm correction in C, D, I, Dunnett's test in E, F against syd-2(lf)]. Scale bars, 10 μm.

Figure 3.

The LH1 domain of SYD-2 is necessary and sufficient for suppressing syd-1(lf) phenotypes. A–E, Activity of pan-neurally expressed SYD-2 transgenic constructs to suppress syd-1(lf) phenotype. A, Localization of synaptic vesicle markers SNB-1::YFP and GFP::RAB-3, presynaptic active zone markers GIT-1::YFP and ELKS-1::GFP at vulval region in wild type, syd-1(ju2), and syd-1(ju2) with SYD-2 transgenes. B, Quantification of SNB-1::YFP localization at HSN synapses. C, Quantification of egg-laying behavior. Values represent percentage of animals (mean ± SD of two independent lines, n = the numbers of animals scored). D, E, Quantification of fluorescence intensity of GFP::RAB-3 (D) and GIT-1::YFP (E) puncta at synaptic region. Values represent relative intensity (mean ± SEM, 5 animals). F, Localization of mCherry::SYD-2 constructs (full-length, N719, LH1, and ΔLH1) at HSN synaptic region in syd-1(ju2). **p < 0.01, *p < 0.05 [Fisher's exact test with Holm correction in B, C, Dunnett's test in D, E against syd-1(ju2)]. Scale bars, 10 μm.

Figure 4.

The LH1 domain of SYD-2/Liprin-α forms a dimer and is important for protein oligomerization and clustering. A, B, In vitro binding of LH1 domains of SYD-2 (LH1) and hLiprin-α1 (hLH1). GST-LH1, GST-LH1Δ (Δ179–198), or GST was pulled down with His6-LH1 (A). GST-hLH1, GST-LH1, or GST was pulled down with His6-hLH1 (B). The numbers indicate the amount of input GST proteins (in micrograms). Eluate and input were analyzed by SDS-PAGE and staining. C, Chemical crosslinking of SYD-2LH1. His6-SYD-2(LH1), SYD-2(1–90), and SYD-2(516–719) were treated with glutaraldehyde, and analyzed by SDS-PAGE and staining. D, Measurement of absolute molecular mass of MBP-LH1 fusion protein. Chromatogram [refractive index (a.u.), black line] is overlaid with calculated molecular mass of the major dimeric peak by MALS (gray line, arrowhead). Arrow indicates peak position corresponding to monomer. E, F, Pan-neural expression of SYD-2-DI in syd-2(ju37) or syd-1(ju2). Quantification of SNB-1::YFP localization (E) and egg-laying behaviors (F). Values represent percentage of animals (mean ± SD of two independent lines, n = the numbers of animals scored). **p < 0.01 (Fisher's exact test with Holm correction). G, BN-PAGE analysis of hLiprin-α1. FLAG-tagged hLiprin-α1 N-terminal proteins [aa 1–520; wild type, R181C, and ΔLH1 (Δ88–214)] were size-separated in native condition by BN-PAGE (top) or in denatured condition by SDS-PAGE (bottom), and analyzed by Western blotting. H–L, Requirement of LH1 domain of hLiprin-α1 on the cluster formation in neuronal cells. GFP-tagged hLiprin-α1 full-length protein (H, wild type, I, R181C, J, ΔLH1) or LH1 domain (K, LH1) was expressed in CAD cells. Representative images of GFP fluorescence in neurites are shown. Scale bar, 20 μm. L, Quantification of total area of Liprin-α clusters in neurites. Values represent Liprin-α cluster index (mean ± SEM of 5–6 individual cells). **p < 0.01, *p < 0.05 (Dunnett's test against wild type).