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Endocytic protein AP180 assembly domain regulates synaptic vesicle size and release in Caenorhabditis elegans [1]

['Yu Wang', 'Fudan University', 'Shanghai', 'P. R. China', 'Basic Sciences Division', 'Fred Hutchinson Cancer Center', 'Seattle', 'Washington', 'United States Of America', 'School Of Life Sciences']

Date: 2026-02 

Neuronal communication relies on neurotransmitter release from synaptic vesicles. The endocytic protein AP180 is critical for efficient vesicle recycling at presynaptic terminals, and its loss impairs neurotransmission, producing reduced release frequency, enlarged synaptic vesicles, and increased quantal amplitude. Yet how AP180 controls vesicle size and whether vesicle size influences release remains unclear. Here, we show that the C-terminal Assembly domain (AD) of AP180 determines vesicle size and thereby regulates release properties in Caenorhabditis elegans. An AP180 variant lacking the AD (AP180∆AD) increases release frequency, contrasting sharply with the reduced transmission in ap180 null mutants, yet fails to correct the vesicle size or quantal amplitude. These enlarged vesicles evade curvature-dependent inhibition by complexin, a presynaptic regulator of fusion, while remaining dependent on complexin for evoked responses. This selective escape reveals that vesicle size influences release dynamics through curvature-sensing proteins. Replacing the AP180 AD with actin-binding motifs restores normal vesicle size, quantal amplitude, and release frequency, indicating that actin interactions are both necessary and sufficient for AD function. Biochemically, we show that the intrinsically disordered AD forms condensates that enrich actin monomers and nucleate filament assembly, while full-length AP180 couples PIP2-rich membranes to actin filaments. Together, these findings reveal that the AP180 AD regulates synaptic vesicle size through actin binding, establishing vesicle morphology as a key influencer of curvature-dependent release control.

Funding: This research was supported by the National Institutes of Health (R01GM127857 to J.B., https://www.nigms.nih.gov ). The Caenorhabditis elegans Genetics Center is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440, https://orip.nih.gov ). The Electron Microscopy Shared Resource at the Fred Hutchinson Cancer Center is supported in part by the NCI Cancer Center Support Grant (P30 CA015704, https://www.cancer.gov ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2026 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we investigated the role of the AP180 AD in C. elegans, uncovering its importance in maintaining synaptic vesicle morphology and safeguarding synaptic transmission. We show that loss of the AD results in enlarged synaptic vesicles and increased quantal amplitudes, resembling the defects observed in ap180 null mutants. Unexpectedly, synapses expressing AP180 lacking the AD exhibit increased, rather than decreased, neurotransmitter release frequency, revealing an inhibitory role of AP180 AD in synaptic transmission. Furthermore, in the absence of AP180 AD, enlarged vesicles evade regulation by complexin, a synaptic protein that senses membrane curvature [ 70 ], revealing an unexpected link between vesicle morphology and neurotransmitter release dynamics. Finally, we show that substituting AP180 AD with actin-binding motifs restores both vesicle size and neurotransmitter release frequency, illustrating a functional link between actin cytoskeleton and AP180 AD in synaptic vesicle regulation.

The endocytic adaptor protein AP180 is central to synaptic vesicle recycling, influencing vesicle morphology and fusogenicity to ensure efficient neurotransmission [ 25 – 27 ]. Loss of AP180 impairs synaptic transmission, disrupts retrieval of synaptic vesicle proteins from the plasma membrane, and leads to abnormally enlarged vesicles in mouse and Drosophila neurons [ 28 – 31 ]. Similarly, mutations in the unc-11 gene, which encodes the Caenorhabditis elegans AP180 ortholog, produce comparable defects, including severely reduced locomotion (likely due to impaired synaptic transmission at neuromuscular junctions), accumulation of synaptic vesicle proteins on the plasma membrane, and enlarged synaptic vesicles [ 32 – 34 ]. These findings demonstrate that AP180 has an essential function in preserving synaptic vesicle quality across animal species. Structurally, AP180 proteins feature a modular architecture, characterized by an N-terminal AP180 N-terminal Homology (ANTH) domain [ 35 , 36 ] and a C-terminal intrinsically disordered Assembly domain (AD) [ 37 ]. The ANTH domain has been extensively studied for its interaction with phosphatidylinositol 4,5-bisphosphate [ 35 , 36 ] and the v-SNARE protein synaptobrevin/VAMP [ 38 ], which mediate membrane binding and vesicle fusion with the plasma membrane [ 39 – 41 ]. By contrast, the intrinsically disordered AD is less well characterized. It is hypothesized to promote membrane curvature during endocytosis through steric pressure arising from its large hydrophobic radius, a property of its disordered nature [ 42 – 46 ]. According to this model, when coupled to the membrane-binding ANTH domain, steric forces generated by the dense packing of multiple AP180 AD copies on membranes induce bending, thereby facilitating vesicle formation.

Endocytosis plays a crucial role in sustaining neuronal communication by replenishing the vesicle pool and preserving vesicle identity and dimensions necessary for precise neurotransmission [ 15 , 18 – 21 ]. A key aspect of endocytosis is the shaping of vesicle dimensions, which directly influences neurotransmitter storage capacity and, consequently, quantal amplitude [ 22 , 23 ]. In addition, endocytosis restores essential proteins and lipids to synaptic vesicles, preserving their identity and ensuring reliable transmission. A hallmark of chemical synapses across animal species is the presence of hundreds to thousands of uniformly-sized vesicles, each about 30–50 nm in diameter [ 18 , 23 – 25 ]. This remarkable uniformity illustrates the conserved ability of endocytosis to reproduce small, consistent vesicle dimensions, suggesting that the mechanisms preserving vesicle size have been evolutionarily maintained.

Neurons communicate through the precisely regulated release of neurotransmitters from synaptic vesicles, small membrane-bound organelles that are the fundamental quanta of synaptic transmission. The concept of quantal neurotransmitter release was first established through electrophysiological recordings at the frog neuromuscular junction [ 1 – 3 ], providing functional evidence for discrete miniature neurotransmission signals. Subsequent ultrastructural analyses revealed a uniform population of synaptic vesicles at presynaptic terminals [ 4 – 13 ], leading to the vesicle hypothesis of synaptic transmission. The demonstration that neurotransmitters are stored within these vesicles [ 14 ] further reinforced their central role in chemical synapses, positioning synaptic vesicles as the morphological correlates of quantal neurotransmission [ 15 – 17 ].

Results

Deletion of unc-11 disrupts synaptic transmission and alters synaptic vesicle dimension in C. elegans To investigate the role of the AP180 ortholog UNC-11 in C. elegans, we used CRISPR-Cas9 to generate the pek217 mutant allele, which removes most of the unc-11 gene, including part of exon 3, exons 4–7, and introns between these exons (S1A Fig). We then performed electrophysiological recordings at neuromuscular junctions in pek217 mutants and compared them to another putative null allele, e47, previously identified in forward genetic screens. The e47 allele lacks 210 base pairs spanning portions of exons 1 and 2 and intron 1 [32,34]. As expected, both pek217 and e47 mutations lead to significant reductions in synaptic transmission (S1B and S1C Fig). Specifically, the amplitude of evoked excitatory postsynaptic currents (evoked EPSCs) is lower in mutants (pek217: 0.20 ± 0.04; e47: 0.19 ± 0.04 nA) compared to wild-type N2 worms (3.24 ± 0.20 nA; p < 0.001, S1B Fig). Similarly, the frequency of endogenous EPSCs is reduced in both pek217 and e47 (pek217: 6 ± 1; e47: 11 ± 3; versus N2: 53 ± 3 Hz; p < 0.001; S1C Fig). In contrast, and consistent with observations in ap180 knockout mice and flies, the amplitude of endogenous EPSCs is elevated in both pek217 (31 ± 2 pA) and e47 (30 ± 2 pA) relative to N2 (22 ± 2 pA; p < 0.001; S1C Fig). Collectively, these data show that unc-11 ap180 is required for normal synaptic transmission in C. elegans, mirroring findings in mice and flies. Because pek217 and e47 mutants displayed similar phenotypes, we used the pek217 allele for subsequent experiments, given its more extensive deletion and lower likelihood of carrying non-specific mutations from chemical mutagenesis. Unless otherwise specified, we refer to pek217 as “unc-11 mutant” in the text and “unc-11 mut.” in the figures.

UNC-11 ∆AD retains activity in synaptic vesicle protein recycling Given that UNC-11∆AD enhances locomotion and synaptic transmission in unc-11 mutants, we hypothesized that synaptic vesicle endocytosis remains functional at UNC-11∆AD synapses. To test this, we tracked synaptic vesicle retrieval in the glutamatergic ASH neuron [41,47]. To visualize endocytosis dynamics, we used a pH-sensitive GFP variant, super-ecliptic pHluorin, inserted into the first lumenal domain of the vesicular glutamate transporter EAT-4 VGLUT, generating a VGLUT-pHluorin reporter (Fig 2A). Under resting conditions, the acidic vesicle lumen quenches VGLUT-pHluorin fluorescence. Upon synaptic vesicle exocytosis, fusion with the plasma membrane exposes VGLUT-pHluorin to the neutral extracellular environment, leading to fluorescence de-quenching and an increase in fluorescence intensity. Subsequent endocytosis and reacidification return the reporter to its quenched state, allowing us to measure vesicle recycling efficiency. As expected, unc-11 ap180 mutants display significantly slower VGLUT-pHluorin retrieval following stimulation (τ = 51 ± 6 s) compared to wild-type worms (19 ± 3 s, Fig 2B and 2D), indicating that synaptic vesicle recycling is impaired in neurons lacking UNC-11. Additionally, baseline VGLUT-pHluorin fluorescence is elevated in unc-11 mutants relative to wild-type worms (Fig 2E), suggesting an accumulation of vesicular proteins at the plasma membrane. These findings are consistent with previous studies showing that AP180 is essential for recycling synaptic vesicle proteins [29,33]. Expression of UNC-11, either full-length or UNC-11∆AD, fully restores vesicle retrieval rates (FL: 25 ± 2 s, and ∆AD: 28 ± 4 s; not significant; Fig 2C and 2D) and normalizes basal VGLUT-pHluorin fluorescence to wild-type levels (Fig 2E). These findings show that removing the AD does not impair the ability of UNC-11 AP180 to facilitate endocytic retrieval of synaptic vesicle proteins from the plasma membrane. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Internalization of synaptic vesicle proteins is preserved in the absence of UNC-11 AD. (A) Schematic of VGLUT-pHluorin assay in ASH neurons. A super-ecliptic pHluorin is inserted into the first lumenal domain of EAT-4 (VGLUT) and expressed under the sar-6p promoter. At rest, fluorescence is quenched by the acidic vesicle lumen. Upon 0.5 M NaCl stimulation (Stimulus On), VGLUT-pHluorin is exposed to the extracellular space via exocytosis, increasing fluorescence. Following stimulus removal (Stimulus Off), vesicle re-acidification quenches the fluorescence as VGLUT-pHluorin is internalized. (B) VGLUT-pHluorin responses over time from wild-type (black) and unc-11 mutant (red) worms. Grey shading indicates stimulus period; SEM is shown. (C) VGLUT-pHluorin responses from wild-type (black) and unc-11 mutant worms expressing either full-length UNC-11 (FL, left, blue) or UNC-11∆AD (right, purple). Summary data of fluorescence recovery rates and basal fluorescence intensity at rest are shown in (D) and (E), respectively. Data are shown as mean ± SEM. n values are indicated on bar graphs. *** p < 0.001 vs. wild type (one-way ANOVA with Tukey’s HSD post hoc test); n.s., not significant. The data underlying this figure are provided in S2 Data. https://doi.org/10.1371/journal.pbio.3003643.g002

Synaptic vesicles remain enlarged despite tethering disordered motifs to UNC-11∆AD to enhance steric pressure To investigate how the AP180 AD controls synaptic vesicle size, we considered a recent model proposing that the AD promotes membrane curvature through steric pressure generated by its large hydrophobic radius [42–46]. In this model, when coupled to the membrane-binding ANTH domain, steric forces generated from densely-packed AP180 AD copies induce membrane bending, facilitating vesicle formation. Supporting this idea, biophysical analyses show that chimeric AP180 proteins, in which the AD was replaced by the C-terminal intrinsically disordered region (CD) of Neurofilament-M (NfM), a protein lacking membrane affinity, retained curvature-inducing activity comparable to that of the native AP180 AD in vitro [45]. These findings suggest a model in which the AD regulates vesicle size through its disordered nature rather than through specific interactions with proteins or membranes. To test whether increasing the hydrophobic radius of disordered motifs could restore synaptic vesicle size in vivo, we generated a chimeric UNC-11 protein by fusing the NfM CD to UNC-11∆AD (Fig 5A), a strategy previously used in vitro [42,45]. Electron microscopy analysis revealed that synaptic vesicles at UNC-11∆AD::NfM-CD synapses remain enlarged (38.8 ± 0.4 nm; Fig 5B and 5C) compared to wild-type (32.1 ± 0.2 nm; p < 0.001), though they are slightly but significantly reduced relative to those in unc-11 mutants (40.2 ± 0.2 nm; p < 0.05) or UNC-11∆AD synapses (40.4 ± 0.2 nm; p < 0.001). Additionally, endosome-like structures appear less frequently at UNC-11∆AD::NfM-CD synapses compared to UNC-11∆AD synapses, though this difference was not statistically significant (Fig 5D, p = 0.14). These results suggest that increasing the hydrophobic radius by tethering disordered motifs provides a modest improvement in synaptic vesicle size and membrane organization at synapses but is not sufficient to fully account for the role of UNC-11/AP180 in synaptic vesicle size control. PPT PowerPoint slide

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TIFF original image Download: Fig 5. C-terminal domains from HIPR-1 and Epsin1 substituted UNC-11 AD for synaptic vesicle (SV) size regulation. (A) Schematics of UNC-11 chimeras in which the UNC-11 ANTH domain (residues 1–304) is fused to the C-terminal intrinsically disordered region of mouse Neurofilament-M (NfM-CD, residues 411–848), the C-terminal domain of Caenorhabditis elegans HIPR-1 (HIPR1-CD, residues 636–928), or the C-terminal domain of rat Epsin1 (Epsin1-CD, residues 316–575). Chimeras were expressed in unc-11 mutant worms via single-copy transgenes driven by the pan-neuronal snb-1p promoter. “NfM,” “HIPR-1,” and “Epsin1” refer to unc-11 mutants expressing each chimera. (B–D) EM analysis of synaptic vesicle dimensions at unc-11 mutant synapses expressing the chimeric UNC-11 proteins. (B) Representative images of transmission electron micrographs for each genotype. Dense projections are labeled in orange. (C) Scatter plot showing the average synaptic vesicle diameter per synaptic profile. Mean values are indicated by the horizontal lines on the graph. Each data point represents one synaptic profile. (D) The fraction of synapses containing ELS is shown in bar graphs for the indicated genotypes. Vesicle diameter data analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test; percentages of synapses with ELS analyzed using Fisher’s exact test. * p < 0.05; *** p < 0.001. The data underlying this figure are provided in S5 Data. https://doi.org/10.1371/journal.pbio.3003643.g005

Tethering HIPR-1 and Epsin1 C-terminal domains to UNC-11∆AD improve vesicle size distribution. To investigate the mechanisms that determine synaptic vesicle size, we considered an alternative model in which the AP180 AD regulates vesicle dimensions through protein interactions shared among other endocytic proteins. This hypothesis was based on three key observations. First, several endocytic proteins, including AP180, huntingtin-interacting-protein-related 1 (HIPR-1), and Epsin1, share a similar domain organization, consisting of an N-terminal membrane-binding ANTH/ENTH domain [35,55,56] and a large C-terminal region [37,57]. Second, these proteins interact [58–60] and function cooperatively to facilitate endocytosis [29,58,61,62]. Third, deletion of individual proteins within this group results in impaired endocytic vesicles in cells [58,63,64]. Supporting this model, we find that deletion of hipr-1, the C. elegans ortholog of yeast Sla2 and human HIP1/HIP1R, results in increased quantal size, elevated endogenous EPSC frequency, and enlarged synaptic vesicles (S5A–S5D Fig). Endosome-like structures were occasionally observed but did not reach statistical significance (S5E Fig). These defects at hipr-1 mutant synapses closely resemble those observed at UNC-11∆AD synapses, suggesting that HIPR-1 and UNC-11 converge on a shared mechanism of vesicle size regulation. These findings prompted us to test whether the C-terminal regions of HIPR-1 and Epsin1 can functionally substitute for the AP180 AD in regulating vesicle size in vivo. To do so, we generated UNC-11∆AD chimeras by fusing UNC-11∆AD to HIPR-1-CD or Epsin1-CD (Fig 5A). Expression of these chimeric proteins in unc-11 mutants significantly reduces synaptic vesicle size (HIPR-1-CD: 35.6 ± 0.5 nm, and Epsin1-CD: 34.7 ± 0.4 nm, respectively) compared to those at UNC-11∆AD synapses or UNC-11∆AD::NfM-CD synapses (Fig 5B-5C). Also, the occurrence of endosome-like structures is lower at UNC-11∆AD::HIPR-1-CD and UNC-11∆AD::Epsin1-CD synapses compared to UNC-11∆AD synapses (Fig 5D). These findings suggest that the C-terminal regions of HIPR-1 and Epsin1 can effectively substitute for the UNC-11 AP180 AD in regulating synaptic vesicle size, supporting the idea that these endocytic proteins contribute to vesicle size regulation through a shared mechanism.

Actin interactions are necessary and sufficient to restore synaptic vesicle size Unlike the UNC-11 AP180 AD, which lacks an ordered three-dimensional structure, the HIPR-1 C-terminal domain contains structured regions, including the highly conserved THATCH (talin/HIP1R/Sla2p actin tethering C-terminal homology) domain, which binds actin (S5A Fig) [65–67]. To determine whether actin binding is involved in vesicle size regulation, we deleted the THATCH domain (∆THATCH) from the UNC-11∆AD::HIPR-1-CD chimera (Fig 6A upper). This deletion disrupts chimera’s ability to restore normal vesicle size, resulting in enlarged synaptic vesicles (39.8 ± 0.3 nm, Fig 6A lower). These findings suggest that actin interactions are required for generating synaptic vesicles of normal size. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Deletion of the actin-binding motif from HIPR-1-CD impairs its ability to substitute for UNC-11 AD in regulating synaptic vesicle size. (A) (Upper) Schematics of UNC-11∆AD::HIPR-1-CD chimeras. The actin-binding THATCH motif is highlighted (yellow). The ∆THATCH variant lacks the C-terminal THATCH domain (deletion of 207 amino acids). Chimeras were expressed in unc-11 mutant worms using a single-copy transgene under the pan-neuronal snb-1p promoter. “∆THATCH” indicates unc-11 mutant worms expressing the ∆THATCH variant. (Lower) Scatter plot of average vesicle diameter per synaptic profile in UNC-11∆AD::HIPR-1-CD and ∆THATCH-rescued unc-11 synapses. Each dot represents a single synaptic profile; horizontal bars indicate mean. (B) (Upper) Schematic of a UNC-11 variant with a 17-aa F-actin binding motif, Lifeact (blue), fused to the C-terminus of UNC-11∆AD (∆AD::Lifeact). The Lifeact amino acid sequence is “MGVADLIKKFESISKEE”. This construct is expressed in unc-11 mutant worms via a single-copy transgene driven by the pan-neuronal snb-1p promoter. (Lower) Scatter plot of average vesicle diameter per profile in ∆AD- and ∆AD::Lifeact-rescued unc-11 synapses. Each dot represents one profile; horizontal bars show means. (C) Excitatory postsynaptic currents (EPSCs) recorded at neuromuscular junctions. Representative traces (upper) and summary data (lower) of endogenous EPSC frequency (left) and amplitude (right) are shown. Data are presented as mean ± SEM. The number of worms analyzed is indicated in the bar graphs. One-way ANOVA followed by Tukey’s HSD post hoc test; * p < 0.05; *** p < 0.001. The data underlying this figure are provided in S6 Data. https://doi.org/10.1371/journal.pbio.3003643.g006 Next, we tested whether enhancing actin binding, without significantly altering the hydrophobic radius, could enable UNC-11∆AD to restore vesicle size. To do this, we fused Lifeact, a 17-amino acid F-actin-binding peptide, to the C-terminus of UNC-11∆AD (Fig 6B upper). Strikingly, adding Lifeact to UNC-11∆AD significantly restores synaptic vesicle size to 35.0 ± 0.3 nm (Fig 6B lower). Functionally, expressing the UNC-11∆AD::Lifeact chimera in unc-11 mutant worms rescues endogenous EPSC frequency (37 ± 5 Hz) and amplitude (26 ± 2 pA) (Fig 6C). These results demonstrate that strengthening actin interactions enables AP180∆AD to regulate synaptic vesicle size, highlighting actin binding as both essential and sufficient for AP180-mediated vesicle size control. To test whether the UNC-11 AD domain can functionally substitute for the HIPR-1 THATCH domain in vivo, we expressed a chimeric HIPR-1 protein in which the THATCH domain was replaced by UNC-11 AD (HIPR-1∆THATCH::AD) in hipr-1 mutant neurons (S5F Fig). This chimera restored endogenous EPSC frequency (56 ± 5 Hz) and amplitude (23.5 ± 1 pA) to wild-type levels (S5G Fig), indicating that the UNC-11 AD domain can replace the actin-binding HIPR-1 THATCH domain in vivo, supporting a model in which both domains share a common mechanistic role in linking endocytosis to the actin cytoskeleton.

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