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The high in vivo stability of 2,2-dihydroxymethyl-3-[18F]fluoropropyl-2-nitroimidazole ([18F]DiFA) prompted us to evaluate neopentyl as a scaffold to prepare a radiotheranostic system with radioiodine and astatine.Three DiFA derivatives with one, two, or without a hydroxyl group were synthesized. While all 125I-labeled compounds remained stable against nucleophilic substitution, only a 125I-labeled neopentyl glycol was stable against cytochrome P450 (CYP)-mediated metabolism and showed high stability against in vivo deiodination. 211At-labeled neopentyl glycol also remained stable against both nucleophilic substitution and CYP-mediated metabolism. 211At labeled neopentyl glycol showed the biodistribution profiles similar to those of its radioiodinated counterpart in contrast to the 125I/211At-labeled benzoate pair. The urine analyses confirmed that 211At-labeled neopentyl glycol was excreted in the urine as a glucuronide conjugate with the absence of free [211At]At-. These findings indicate that neopentyl glycol would constitute a promising scaffold to prepare a radiotheranostic system with radioiodine and 211At.

As synaptic vesicles fuse, they must continually be replaced with new docked, fusion-competent vesicles to sustain neurotransmission. It has long been appreciated that vesicles are recruited to docking sites in an activity-dependent manner. However, once entering the sites, vesicles were thought to be stably docked, awaiting calcium signals. Based on recent data from electrophysiology, electron microscopy, biochemistry, and computer simulations, a picture emerges in which vesicles can rapidly and reversibly transit between docking and undocking during activity. This "transient docking" can account for many aspects of synaptic physiology. In this review, we cover recent evidence for transient docking, physiological processes at the synapse that it may support, and progress on the underlying mechanisms. We also discuss an open question: what determines for how long and whether vesicles stay docked, or eventually undock?

Targeted α-radionuclide therapy has growing attention as a promising therapy for refractory cancers. However, the application is limited to certain types of cancer. Since L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, we prepared LAT1-selective α-emitting amino acid analog, 2-[211At]astato-α-methyl-L-phenylalanine (2-[211At]AAMP), and evaluated its potential as a therapeutic agent. Methods: 2-[211At]AAMP was prepared from the stannyl precursor. Stability of 2-[211At]AAMP was evaluated by both in vitro and in vivo. In vitro studies using LAT1 expressing human ovarian cancer cell line, SKOV3, were performed for evaluating cellular uptake and cytotoxicity of 2-[211At]AAMP. Biodistribution and therapeutic studies in SKOV3 bearing mice were performed after intravenous injection of 2-[211At]AAMP. Results: 2-[211At]AAMP was stable in murine plasma in vitro and excreted into urine as intact. Cellular uptake of 2-[211At]AAMP was inhibited by treatment with LAT1-selective inhibitor. After 24 hours of incubation, 2-[211At]AAMP suppressed clonogenic growth at 10 kBq/ml, and induced cell death and DNA double-strand break at 25 kBq/ml. When injected to mice, 2-[211At]AAMP exhibited the peak accumulation in the tumor at 30 min postinjection, and the radioactivity levels in the tumor retained up to 60 min. The majority of the radioactivity was rapidly eliminated from the body into the urine as an intact form immediately after injection. 2-[211At]AAMP significantly improved the survival of mice (P

Objective We aimed to estimate in vivo 211At-labeled meta-benzylguanidine (211At-MABG) absorbed doses by the two dose conversion methods, using 131I-MIBG biodistribution data from a previously reported neuroblastoma xenograft model. Additionally, we examined the effects of different cell lines and time limitations using data from two other works. Methods We used the framework of the Monte Carlo method to create 3200 virtual experimental data sets of activity concentrations (kBq/g) to get the statistical information. Time activity concentration curves were produced using the fitting method of a genetic algorithm. The basic method was that absorbed doses of 211At-MABG were calculated based on the medical internal radiation dose formalism with the conversion of the physical half-life time of 131I to that of 211At. We have further improved the basic method, that is a novel dose conversion method, RAP (RAtio of Pharmacokinetics), using percent injected dose/g. Results Virtual experiments showed that 211At-MABG and 131I-MIBG had similar properties of initial activity concentrations and biological components, but the basic method did not simulate the 211At-MABG dose. Simulated 211At-MABG doses from 131I-MIBG using the RAP method were in agreement with those from 211At-MABG, so that their boxes overlapped in the box plots. The RAP method showed applicability to the different cell lines, but it was difficult to predict long-term doses from short-term experimental data. Conclusions The present RAP dose conversion method could estimate 211At-MABG absorbed doses from the pharmacokinetics of 131I-MIBG with some limitations. The RAP method would be applicable to a large number of subjects for targeted nuclide therapy.

This study demonstrated the availability of an anion-exchange spin column method for the isolation of astatine-211 (At-211) from bismuth (Bi). At-211 was isolated in medium-to-excellent recovery efficiencies when loading At-211 with 8M HCl (75.3–79.1%), and 8M HNO3 (35.9–54.3%). The processing time of the optimized protocol was about 40 minutes. A neutralized eluent afforded an 211At-labeled phenylalanine derivative even in the absence of oxidants (radiochemical yield: 75.4%). These results indicate the anion-exchange spin column is useful for the isolation of At-211 and the following synthesis of astatinated compounds, which is the first to isolate At-211 with an anion-exchange method.

211At was produced in the 209Bi(4He, 2n)211At reaction. The yield was separated from the irradiated bismuth target by a simple method, based on dry distillation. The optimized conditions of the method were studied by monitoring the astatine radioactivity with gamma-ray spectrometers (GRSs). The no-carrier-added 211At solutions were prepared with eluents, namely distilled water, ethanol, methanol, or chloroform. Generally, the recovery yields obtained with the utilization of 1.6 mL of the eluent were 26-75%. The prepared 211At solutions were subjected to thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to identify the chemical species present.

Simple Summary To enhance the therapeutic effect of 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), a radiopharmaceutical for targeted alpha therapy, we evaluated the effect of probenecid loading on its biodistribution and therapeutic effect in mice. Probenecid preloading significantly delayed the clearance of 2-211At-AAMP from the blood, increasing its accumulation in tumors. Consequently, the therapeutic effect of 2-211At-AAMP markedly improved. These results indicate that 2-211At-AAMP with probenecid loading is useful for the treatment of various types of cancers. Abstract L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-211At-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-211At-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-211At-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-211At-AAMP in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-211At-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-211At-AAMP by increasing its accumulation in tumors.

Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently., Action potentials induce synchronous and asynchronous release of neurotransmitters. Here, the authors show that the synchronous and asynchronous release sites are aligned with AMPARs and NMDARs, respectively, in mouse hippocampal synapses. This organization allows efficient activation of NMDARs.

Astatine-211 (211At) is one of the most promising radionuclides for targeted α therapy of cancers and radioiodine such as iodine-123 is useful radionuclide for diagnosis. Because astatine and iodine are the same group, halogen, the same scaffolds could be used for astatination and radioiodination to prepare radiotheranostics compounds. However, the in vivo instability of 211At-labeling agents such as 211At-benzoate limits the application of 211At to various compounds. To overcome the problem and expand the application of 211At-labeled compounds to radiotheranostics, we developed neopentyl derivatives as a novel scaffold for astatination and radioiodination. The stability and biodistribution of 211At or 125I-labeled neopentyl derivatives were compared with reference compounds radiolabeled with 125I or 211At via a conventional method using benzamide., SNMMI2020 Annual Meeting

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

Astatine-211 is one of the promising radioisotopes for targeted alpha therapy. Optimising treatment strategies as well as determining the suitability of a given agent for a particular patient requires to image the time-dependent distribution of the targeted radiotherapeutic agent both in tumours and in normal tissues. Since the biodistribution of astatine is different from that of iodine, imaging astatine-211 directly is essential. In the previous study of astatine-211 Compton imaging, random coincidence events due to polonium K-shell X-rays were dominant and seemed to cause saturation of counts. Thus optimisation of the coincidence time windows is important to reduce random coincidence events. In this study, we have optimised the coincidence time windows of a Compton camera and improved the sensitivity, noise and spatial resolution of astatine-211 imaging.

Astatine-211 is one of the promising alpha-emitting radionuclides for the targeted alpha therapy (TAT), internal radiotherapy using alpha-emitting radioisotopes. At-211 is basically produced by irradiating He beams with 28–29 MeV to a bismuth (Bi) target. Irradiations with high beam current (µA order) are necessary to produce At-211 for preclinical and clinical studies because they require its radioactivity on the order of subGBq to GBq. Low melting point of Bi (273.1°C) often causes melting of the Bi during irradiation with high beam current. The aim of this study is therefore to optimize irradiation condition without observing Bi melting.

Case The standard treatment for acute subdural hematoma (ASDH) is large craniotomy; decompressive craniectomy may also be carried out, if needed, to prevent secondary brain damage. Recently, an endoscopic procedure for elderly patients with ASDH was carried out and reported; its safety and effectiveness were emphasized because of minimal invasiveness. We report a young case and discuss its difficulties and tips. A 31-year-old man was found to be in a state of general convulsion. At the time of admission, we observed severe consciousness disturbance, anisocoria, and left hemiparesis. Computed tomography showed a massive subdural hematoma with marked midline shift. Outcome Osmotherapy and emergency trepanation improved anisocoria. An endoscopic procedure under local anesthesia was sequentially selected. After surgery, the patient's symptoms clearly improved. Conclusion Although the standard treatment for ASDH is craniotomy, endoscopic surgery may be useful in some cases.

We discuss a novel broadband, polarization-insensitive, and wavelength-shift-free optical phase conjugator (OPC). In the OPC scheme, the original input signal is inherently suppressed in a polarization-diversity loop setup. This allows for spectrally efficient, wavelength-(band)-shift-free generation of phase-conjugated idlers over a continuous operating band. The idlers are generated using four-wave mixing in nonlinear fibers using out-of-band, orthogonally polarized pump waves. The saturation characteristics of the exploited nonlinear mixing process as well as the crosstalk characteristics of the OPC diversity loop scheme are investigated. The OPC subsystem is finally utilized for fiber nonlinearity mitigation by midlink spectral inversion in 800-km transmission experiments over dispersion-managed fiber spans with lumped amplification by Erbium-doped fiber amplifiers. Q 2-factor improvements of up to 0.7 dB are obtained in transmission of a 300-GHz wide 1.6 Tb/s super channel consisting of eight 200 Gb/s polarization division multiplexed 16-ary quadrature amplitude modulation subchannels.

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here, we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans. Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq-Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans. Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

We propose an optical frequency shifter for dual-polarization signals using frequency conversion in fiber. The proposed optical frequency shifter is achieved by transmitting input signal combined with two orthogonally polarized continuous wave pump lights through a nonlinear fiber and a polarizer. In the scheme, a polarization switched and frequency shifted copy of an original signal is generated in the nonlinear fiber and the subsequent polarizer removes the original signal in a wavelength independent fashion. It allows a fractional bandwidth shift, in which the shifted signal is overlapped with the original signal. We achieve THz range frequency shift with original signal suppression without an optical filter by using a polarization diversity loop. A polarization beam splitter is used as a splitter and a combiner of the loop as well as for original signal rejection. A contention resolution of two dual-polarization 400-Gb/s superchannels is demonstrated by applying the optical frequency shifter with acceptable in-band crosstalk. For any-to-any optical frequency shift, wavelength arrangement for pumps and required zero dispersion and dispersion slope of nonlinear fibers are numerically simulated.

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

著者人数: 16名, 資料番号: SA1004683000

We propose an extended coherent optical add/drop multiplexing scheme, in which an individual polarization tributary in a polarization-division multiplexed orthogonal frequency-division multiplexed signal is erased and added by frequency conversion in fiber, which is sequentially inserted in a transmission line. In the scheme, a polarization tributary can be inserted and replaced in a dual polarization spectrally overlapped ultra-dense multiplexed signal. In a proof-of-concept demonstration, we investigate the performance of the proposed optical add/drop multiplexing scheme. A 25-Gb/s quadrature phase-shift keying tributary within a dual-polarization three subcarrier orthogonal frequency division multiplexed signal is erased and added with negligible distortion of the orthogonal polarization tributary and the adjacent subcarriers.

Recently developed upgrade options for distributed ultra-dense frequency-division multiplexing toward large aggregation capacity are discussed. The concept, originally demonstrated for distributed coherent optical orthogonal frequency-division multiplexing, is based on optical frequency conversion in fiber. It enables precise frequency allocation of the successively multiplexed subcarriers within the super-channel without the need of absolute optical frequency control, i.e., allowing for use of free-running lasers at the individual multiplexing nodes. Follow-up experiments proved the applicability of this concept also to Nyquist wavelength-division multiplexing (WDM) with extremely small guard bands by successfully demonstrating the distributed aggregation of a spectrally efficient 400-Gb/s single-polarization zero-guard-band Nyquist-WDM super-channel using 4× 28-GBd 16-ary quadrature-amplitude modulation (16QAM) subcarriers. Recently, we further extended the concept to distributed generation of dual-polarization super-channels. This extension allowed for the distributed aggregation of a 400-Gb/s Nyquist-WDM super-channel using 4× 28-GBd polarization-division multiplexed quaternary phase-shift keying (PDM-QPSK) subcarriers. In this invited contribution, we review and compare in detail both experiments enabling 400-Gb/s aggregation capacity. Additionally, we discuss a viable option for operation under random fiber birefringence regardless of the polarization-dependent nature of the underlying optical frequency conversion process.

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

We demonstrate the use of frequency conversion in optical fiber to implement a coherent add/drop architecture. Its application is experimentally investigated in an optical frequency-division multiplexing scenario with up to three subcarriers which are modulated by quadrature phase-shift keying. We show sub-carrier drop and replacement in a bus topology. Furthermore, we quantify tolerances with respect to delay and amplitude mismatch by experiments and simulations. Finally, we discuss on the scalability of the scheme.

Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50–100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 s, which in turn become small-diameter synaptic vesicles 5–6 s after stimulation. We disrupted clathrin function using RNA interference (RNAi) and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results may explain discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis. Ultrastructural analysis of synaptic vesicle recycling reveals that clathrin is not required for the initial rapid step of vesicle recycling by ultrafast endocytosis at the plasma membrane and instead clathrin acts later at an endosome to regenerate synaptic vesicles; however, when ultrafast endocytosis does not occur (for example, in experiments at room temperature rather than physiological temperature), clathrin-mediated endocytosis does happen at the plasma membrane. Since the discovery of synaptic vesicle recycling in the 1970s, clathrin has been thought to act at the plasma membrane to reconstitute synaptic vesicles about 20 seconds after stimulation of the axon terminal. Erik Jorgensen and colleagues have revisited these original experiments, but combining optogenetic stimulation with high-pressure freezing to rapidly fix the samples after stimulation. This changes the picture: the previously hypothesized mechanism operates at a non-physiological, 'room temperature' of 22°C, when ultra-fast endocytosis is disrupted. At a physiological temperature (34°C), large vesicles are instead retrieved from the plasma membrane within about 50 milliseconds, through clathrin-independent, ultra-fast endocytosis; these then fuse to form endosomes from which small synaptic vesicles are regenerated by clathrin scaffolds, about 5 seconds after stimulation.

Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI: http://dx.doi.org/10.7554/eLife.00723.001, eLife digest Neurons communicate with one another at junctions called synapses. When an electrical signal travels along a neuron and arrives at a synapse, vesicles filled with small neurotransmitter molecules fuse with the cell membrane and release the neurotransmitter. These chemicals rapidly bind to receptors on the downstream neuron that induce an electrical response in that cell. Vesicles can be consumed at prodigious rates, up to 500 a second, so the cell must recover the membrane rapidly and regenerate more vesicles filled with neurotransmitter. Experiments in the 1970s and 1980s suggested that when vesicles empty their contents into the synapse, they fuse completely with the membrane and are lost. To recover the membrane, the cell forms ‘pits’, by means of a coat protein called clathrin, which then bud off into the cell as new vesicles. It takes roughly 15–20 s for vesicles to be recycled in this way. By contrast, synapses with very high firing rates are thought to recycle vesicles through a faster process known as ‘kiss and run’, in which vesicles are not fully integrated into the membrane, but instead fuse transiently with it to form a reversible pore within about a second. However, these studies triggered vesicle release using conditions that are unlikely to occur naturally inside cells. Now, Watanabe et al. have used optogenetics to study vesicle recycling in response to single stimuli at the synapse between neurons and muscles in an intact living animal, the nematode C. elegans. The worms had been genetically modified to express a light-sensitive ion channel called channelrhodopsin in their motor neurons. Watanabe et al. used a single pulse of light to stimulate vesicle release, and then rapidly froze the worms before studying their synapses with electron microscopy. They found that vesicle recycling occurred at the edges of the synapse or at a specialized structure in the middle of the synapse. Vesicle recycling took less than 50 ms—much faster than anything previously observed. This ultrafast recycling is unlikely to occur via ‘kiss and run’ since recycling occurred at sites lateral to the sites of fusion and because the recycled vesicles were larger than the originals, implying that they had not simply re-formed after a brief fusion event. By using physiologically relevant stimuli in an intact animal, Watanabe et al. reveal that vesicles can be recycled at synapses much more rapidly than previously thought, suggesting that our current models of this process may need to be reassessed. DOI: http://dx.doi.org/10.7554/eLife.00723.002

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001, eLife digest The cell membrane is a busy place, with cell-surface proteins continually added and removed according to the needs of the cell. Each protein extends a polypeptide tail into the cell cytoplasm. When a protein is to be removed from the cell surface, its tail recruits a protein complex known as the AP2 adaptor to the membrane. AP2 then recruits a coat protein called clathrin, which forms a spherical scaffold around the adaptor, the target protein and the surrounding membrane, enclosing them inside a vesicle that breaks off from the membrane and enters the cell. Endocytosis is particularly common in neurons, which use it as a means of recycling proteins at synapses—the contact points between nerve cells. However, it is unclear whether synaptic-vesicle recycling also involves clathrin and AP2. To address this question, Gu et al. examined mutant nematode worms (C. elegans) in which the composition of AP2 had been altered. AP2 has four subunits, called α, β2, μ2 and σ2, and Gu et al. produced worms that lack either the α- or μ2-subunit, or both. Few worms that lacked both subunits survived. Surprisingly, however, worms that lacked just one subunit were viable, despite previous evidence that AP2 requires all four subunits to be functional. Nevertheless, these single mutants produced 30% fewer synaptic vesicles compared to wild-type worms. To examine the consequences of both subunits being absent, Gu et al. rescued the double mutants by selectively expressing AP2 in their skin. These animals—which still lack AP2 in their nervous systems—produced 70% fewer synaptic vesicles than their wild-type counterparts. The results show that AP2 does not need all four of its subunits and that it can exist as two semi-independent hemicomplexes. Moreover, Gu et al. show that C. elegans uses at least two endocytotic mechanisms (AP2-dependent and independent) to recycle vesicles and so maintain synaptic function. DOI: http://dx.doi.org/10.7554/eLife.00190.002