Title: Author response: Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses. Editor's evaluation Neuronal function requires the recycling of synaptic vesicle proteins from the presynaptic plasma membrane to reform synaptic vesicles. This study highlights a clathrin-independent role of the clathrin adaptor, AP-2 in the endocytic retrieval of select synaptic vesicle cargos at mammalian central synapses. The work will be of interest to cell biologists and neurobiologists interested in understanding the molecular basis of neurotransmission. https://doi.org/10.7554/eLife.71198.sa0 Decision letter eLife's review process Introduction Synaptic transmission relies on the release of neurotransmitters by calcium-triggered exocytic fusion of synaptic vesicles (SVs), tiny organelles (~40 nm in diameter) that store and secrete neurotransmitter molecules at specialized active zone (AZ) release sites within presynaptic nerve terminals (Südhof, 2004). Following exocytosis, SVs are locally reformed via compensatory endocytic retrieval of membrane and its protein constituents (i.e., SV proteins) from the cell surface in order to keep the presynaptic surface area constant and to ensure sustained neurotransmission (Dittman and Ryan, 2009; Kononenko and Haucke, 2015). While the core components that mediate the exo- and endocytosis of SVs have been identified and characterized in detail (Rizzoli, 2014; Takamori et al., 2006; Wilhelm et al., 2014), the molecular mechanisms underlying SV endocytosis and reformation have remained controversial (Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017; Watanabe et al., 2013; Watanabe et al., 2014; Milosevic, 2018). Pioneering ultrastructural analyses of stimulated frog neuromuscular junctions using electron microscopy (EM) suggested that SVs are recycled via clathrin-mediated endocytosis (CME) of plasma membrane infoldings or budding from cisternal structures located away from the AZ (Heuser and Reese, 1973). Subsequent studies in neurons and in nonneuronal models showed that CME occurs on a timescale of many seconds and crucially depends on clathrin and its essential adaptor protein complex 2 (AP-2) (Mitsunari et al., 2005), a heterotetramer comprising α, β2, μ2, and σ2 subunits, as well as a plethora of endocytic accessory proteins (Südhof, 2004; Dittman and Ryan, 2009; Kononenko and Haucke, 2015; Rizzoli, 2014; Saheki and De Camilli, 2012; Kaksonen and Roux, 2018). These and other works led to the view that SVs are primarily, if not exclusively, recycled by clathrin/AP-2-dependent CME (Granseth et al., 2006; Granseth et al., 2007). Recent studies using high-pressure freezing EM paired with optogenetic stimulation have unraveled a clathrin-independent mechanism of SV endocytosis (CIE) in response to single action potential (AP) stimuli that selectively operates at physiological temperature (Watanabe et al., 2013). This ultrafast endocytosis (UFE) pathway is distinct from kiss-and-run or kiss-and-stay exoendocytosis observed in neuroendocrine cells (Alés et al., 1999; Shin et al., 2018), operates on a timescale of hundreds of milliseconds, and results in the generation of endosome-like vacuoles (ELVs) from which SVs can reform via clathrin-mediated budding processes (Kononenko et al., 2014; Watanabe et al., 2014). Temperature-sensitive, clathrin-independent SV endocytosis has also been observed by presynaptic capacitance recordings at cerebellar mossy fiber boutons and by optical imaging at small hippocampal synapses (Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017) and is compatible with the accumulation of postendocytic presynaptic vacuoles upon acute or sustained genetic perturbation of clathrin at stimulated fly neuromuscular junctions (Kasprowicz et al., 2014; Kasprowicz et al., 2008; Heerssen et al., 2008) and at mammalian central synapses (Kononenko et al., 2014; Imig et al., 2020). Collectively, these studies suggest that SV endocytosis under physiological conditions is primarily mediated by CIE (e.g., UFE), while the function of clathrin and clathrin adaptors such as AP-2 is limited to the reformation of functional SVs from internal ELVs rather than acting at the plasma membrane proper. While this model can provide a mechanistic explanation for the observed speed of SV endocytosis, the key question of how SV proteins are sorted to preserve the compositional integrity of SVs (Takamori et al., 2006) remains unresolved. Four points pertaining to this question are to be considered. First, optical imaging-based acid quench experiments in hippocampal neurons indicate that the capacity of UFE is limited to single or few APs, while the majority of SV proteins appear to be internalized on a timescale of several seconds following AP train stimulation (Dittman and Ryan, 2009; Soykan et al., 2017; Kim and Ryan, 2009), that is a timescale compatible with either CME or CIE. Second, as high-pressure freezing EM experiments have not been able to reveal the fate and time course of endocytosis of SV proteins, it is formally possible that UFE proceeds under conditions of clathrin depletion (Watanabe et al., 2014), while SV proteins remain stranded on the neuronal surface. Third, mutational inactivation of the binding motifs for the clathrin adaptor AP-2 in the vesicular transporters for glutamate (VGLUT) and γ-aminobutyric acid (VGAT) severely compromises the speed and efficacy of their endocytic retrieval at room temperature (Voglmaier et al., 2006; Santos et al., 2013; Li et al., 2017; Foss et al., 2013), arguing that at least under these conditions (e.g., low temperature when UFE is blocked) these SV proteins may be retrieved from the cell surface via clathrin/AP-2. Finally, genetic inactivation of clathrin/AP-2-associated endocytic adaptors for the sorting of specific SV proteins, for example Stonin 2, an adaptor for the SV calcium sensor Synaptotagmin, or AP180, an adaptor for Synaptobrevin/VAMP2, causes the accumulation of their respective SV cargos at the neuronal plasma membrane (Kononenko and Haucke, 2015; Cousin, 2017; Kaempf et al., 2015; Koo et al., 2015; Mori and Takamori, 2017). These data could be interpreted to indicate that at least some SV proteins are endocytosed via CME, whereas others may use CIE mechanisms such as UFE. However, such a model bears the problem of how CME and CIE pathways are coordinated and how membrane homeostasis is then maintained. To solve the question how SV protein sorting is accomplished and how this relates to CME- vs CIE-based mechanisms for SV endocytosis, we have conducted a comprehensive survey of six major SV proteins in primary hippocampal neurons depleted of clathrin or conditionally lacking AP-2. We show that clathrin is dispensable for the endocytosis of all SV proteins at physiological temperature independent of the stimulation paradigm. In contrast, endocytic retrieval of a subset of SV proteins including VGLUT1 and VGAT depends on sorting by AP-2. Our findings highlight a clathrin-independent function of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic plasma membrane and suggest a revised model for SV endocytosis and recycling. Results Based on prior works (Dittman and Ryan, 2009; Rizzoli, 2014; Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017; Watanabe et al., 2014; Milosevic, 2018; Heuser and Reese, 1973; Saheki and De Camilli, 2012; Takei et al., 1996), three main models for the sorting and endocytic recycling of SV proteins at central mammalian synapses can be envisaged (Figure 1). According to the classical CME-based model of SV endocytosis, SV proteins exocytosed in response to AP trains undergo clathrin/AP-2-mediated sorting and endocytosis from the presynaptic plasma membrane or plasma membrane infoldings (Takei et al., 1996) akin to CME in receptor-mediated endocytosis in nonneuronal cells (Kaksonen and Roux, 2018). This model predicts that loss of either clathrin or its essential adaptor AP-2 delays the endocytic retrieval of all major SV proteins (Figure 1A). A second model supported by elegant high-pressure freezing (Watanabe et al., 2014), electrophysiological (Delvendahl et al., 2016), and optical imaging (Kononenko et al., 2014; Soykan et al., 2017) experiments suggests that exocytosed SV proteins are internalized via clathrin- and AP-2-independent bulk endocytosis. In this model, SV protein sorting occurs from internal ELVs that are formed downstream of the endocytic internalization step. Hence, at physiological temperature the endocytic retrieval of all major SV proteins would proceed unperturbed in the absence of either clathrin or AP-2 (Figure 1B). Finally, it is conceivable that exocytosed SV proteins present on the neuronal surface are sorted by dedicated endocytic adaptors, for example the AP-2 complex, to facilitate their clathrin-independent internalization via CIE. Clathrin, possibly in conjunction with AP-2 and other adaptors then operates downstream of CIE to reform functional SVs from ELVs. In this case, loss of clathrin or AP-2 is predicted to result in distinct phenotypes: While endocytosis of SV proteins is unperturbed upon depletion of clathrin, loss of AP-2 would be expected to selectively affect the rate and efficacy of endocytosis of distinct SV cargos recognized by AP-2 (Figure 1C). Figure 1 Download asset Open asset Possible roles of clathrin and adaptor protein complex 2 (AP-2) in synaptic vesicle (SV) endocytosis and SV cargo retrieval. (A) A model predicting that SV retrieval following neurotransmitter release is mediated by clathrin-mediated endocytosis (CME) where AP-2 functions as bridge between SV cargos and clathrin to form clathrin-coated pits (CCPs) at plasma membrane. In this scenario, inactivation of both clathrin and AP-2 would slow either SV endocytosis as well as SV cargo retrieval. (B) A model predicting that SV endocytosis occurs in a clathrin-independent manner (CIE), and neither clathrin nor AP-2 mediate SV endocytosis and SV cargo retrieval at plasma membrane. If this were the case, inactivation of both clathrin and AP-2 would not change the kinetics rate of SV endocytosis and SV cargo retrieval. (C) A model predicting that dedicated adaptors such as AP-2 function as sorting protein for SV cargo even during CIE. If this were the case, inactivation of clathrin and AP-2 would produce distinct phenotypes between SV endocytosis and SV cargo retrieval. Endocytic retrieval of SV proteins in hippocampal neurons occurs independent of clathrin at physiological temperature To distinguish between these models, we optically recorded the stimulation-induced exo-/endocytosis of SV proteins carrying within their luminal domains a pH-sensitive superecliptic green fluorescent protein (SEP, often also referred to as pHluorin) that is dequenched during exocytosis and undergoes requenching as SVs are internalized and reacidified (Miesenböck et al., 1998; Sankaranarayanan et al., 2000). Specifically, we monitored SEP-tagged chimeras of the calcium sensor Synaptotagmin 1 (Syt1), the multispanning glycoprotein SV2A, the SNARE protein Synaptobrevin/VAMP2 (hereafter referred to as Syb2), the tetraspanin Synaptophysin (Syp), the vesicular glutamate transporter 1 (VGLUT1), and the vesicular GABA transporter (VGAT), which have been used extensively to monitor SV recycling in various preparations (Kononenko et al., 2014; Soykan et al., 2017; Granseth et al., 2006; Kim and Ryan, 2009; Voglmaier et al., 2006; Miesenböck et al., 1998; Sankaranarayanan et al., 2000) and constitute the major protein complement of SVs based on their copy numbers (Takamori et al., 2006). We capitalized on the fact that, in hippocampal neurons stimulated with trains of APs, SV endocytosis occurs on a timescale of >10 s at physiological temperature (Soykan et al., 2017), for example a timescale that is much slower than requenching of SEP due to reacidification of newly endocytosed vesicles (Atluri and Ryan, 2006; Egashira et al., 2015). Therefore, under these conditions, the decay of SEP signals can serve as a measure of the time course of SV endocytosis. We first depleted clathrin heavy chain (CHC) in hippocampal neurons using lentiviral vectors to ~10–25% of the levels found in controls as evidenced by confocal imaging of immunostained samples and by immunoblot analysis (Figure 2—figure supplement 1A–D), in agreement with previous data (Kononenko et al., 2014; Watanabe et al., 2014; Granseth et al., 2006). Lentiviral shRNA-mediated depletion of clathrin potently inhibited uptake of transferrin into cultured neurons, indicating effective blockade of CME (Figure 2A, B). To assess the effects of clathrin loss on the stimulation-induced endocytic retrieval of SV proteins, we stimulated control or clathrin-depleted hippocampal neurons expressing any one of the six major SEP-tagged SV proteins with a high-frequency stimulus train (200 APs applied at 40 Hz) at physiological temperature (35 ± 2°C), and monitored fluorescence rise and decay over time. Strikingly, exo-/endocytosis of all SEP-tagged SV proteins proceeded with unaltered kinetics, that is τ ~ 15–20 s, irrespective of the depletion of clathrin (Figure 2C–N). Similar results were seen if clathrin function was acutely blocked by application of the small molecule inhibitor Pitstop2 (von Kleist et al., 2011; Figure 2O, P), a condition that potently inhibited CME of transferrin (Figure 2—figure supplement 1E,F). When these experiments were repeated under conditions of low-frequency stimulation (200 APs applied at 5 Hz) at room temperature (RT), that is conditions in which the efficacy of CIE is reduced (Watanabe et al., 2014), the endocytic retrieval of Syp-SEP (also often referred to as SypHy) or VGLUT1-SEP was delayed in neurons depleted of clathrin (Figure 2—figure supplement 1G-J), consistent with earlier data using Syt1-SEP as a reporter (Kononenko et al., 2014). Figure 2 with 1 supplement see all Download asset Open asset Synaptic vesicle (SV) endocytosis in hippocampal neurons occurs independent of clathrin at physiological temperature. (A) Representative images of primary neurons transduced with shCTRL or shCHC and allowed to internalize AlexaFluor647-labeled transferrin (Tf647) for 20 min at 37°C. The scale bar represents 20 μm. (B) Quantification of data shown in (A). Values for shCTRL were set to 1. The data represent mean ± standard error of the mean (SEM) from n = 6 independent experiments. ****p = 0.0001, two-sided one-sample t-test. (C–N) Average normalized SEP fluorescence traces of neurons transduced with lentivirus expressing nonspecific shRNA (shCTRL) or shRNA-targeting CHC (shCHC) and cotransfected with SEP probes tagged to the luminal portion of Syt1 (C), SV2A (E), VGLUT1 (G), VGAT (I), Syp (K), and Syb2 (M) subjected to electrical stimulation of 40 Hz (200 APs) at physiological temperature. Endocytic decay time constant (τ) of transfected and lentivirally transduced neurons coexpressing, respectively, (D) Syt1-SEP and shCTRL (15.22 ± 1.30 s) or shCHC (14.87 ± 1.87 s); (F) SV2A-SEP and shCTRL (20.00 ± 3.53 s) or shCHC (23.06 ± 3.31 s); (H) VGLUT1-SEP and shCTRL (17.67 ± 2.05 s) or shCHC (20.79 ± 4.07 s); (J) VGAT-SEP and shCTRL (15.82 ± 2.04 s) or shCHC (14.38 ± 1.14 s); (L) Syp-SEP and shCTRL (18.65 ± 2.03 s) or shCHC (21.27 ± 3.10 s); and (N) Syb2-SEP and shCTRL (50.63 ± 7.49 s) or shCHC (42.32 ± 8.03 s). Data shown represent the mean ± SEM for Syt1 (nCTRL = 19 images, nshCHC = 13 images; p = 0.875), for SV2A (nshCTRL = 14 images, nshCHC = 17 images; p = 0.533), for VGLUT1 (nshCTRL = 7 images, nshCHC = 7 images; p = 0.506), for VGAT (nshCTRL = 13 images, nshCHC = 14 images; p = 0.534), for Syp (nshCTRL = 8 images, nshCHC = 8 images; p = 0.490), and for Syb2 (nshCTRL = 17 images, nshCHC = 15 images; p = 0.455). Two-sided unpaired t-test. (O, P) Endocytosis of VGAT upon acute inactivation of clathrin by Pitstop2 proceeds unaffected at physiological temperature. (O) Average normalized traces of neurons transfected with VGAT-SEP and treated either with DMSO (CTRL) or Pitstop2 in response to 200 APs applied at 40 Hz. (P) Endocytic decay time constant (τ) of neurons expressing VGAT-SEP (τCTRL = 14.03 ± 1.16 s, τPitstop2 = 18.11 ± 2.32 s). Data shown represent the mean ± SEM with n = 18 images and n = 13 images for CTRL and Pitstop2, respectively. p = 0.0976. Two-sided unpaired t-test. Raw data can be found in Figure 2—source data 1. Figure 2—source data 1 Source data for Figure 2B-P. https://cdn.elifesciences.org/articles/71198/elife-71198-fig2-data1-v1.xlsx Download elife-71198-fig2-data1-v1.xlsx These results show that in small hippocampal synapses at physiological temperature, endocytosis of all major SV proteins and hence, of SVs as a whole, occurs independent of clathrin via CIE. CIE of a subset of SV proteins depends on the clathrin adaptor AP-2 We next set out to analyze whether endocytosis of the major SV proteins is also independent of the essential clathrin adaptor complex AP-2. This would be expected, if SV endocytosis was mediated by CIE and the sole function of clathrin/AP-2 was to reform SVs from postendocytic ELVs (Figure 1B). We conditionally ablated AP-2 expression by tamoxifen induction of Cre recombinase in hippocampal neurons from Ap2m1lox/lox mice crossed with inducible CAG-Cre transgenic mice resulting in a reduction of AP-2 levels to <15% of that detected in WT control neurons (hereafter referred to as AP-2µ KO) (Figure 3—figure supplement 1A,B; Kononenko et al., 2014; Soykan et al., 2017). Further depletion below this level caused neuronal death. Endocytosis of Syt1-SEP and SV2A-SEP proceeded with similar kinetics in control or AP-2μ KO hippocampal neurons stimulated with 200 APs applied at 40 Hz at physiological temperature, consistent with our earlier findings (Kononenko et al., 2014; Soykan et al., 2017; Figure 3A–D). Surprisingly, however, we found that loss of AP-2 significantly slowed down the endocytic retrieval of other major SV proteins such as Syp, Syb2, and most prominently, of the vesicular neurotransmitter transporters VGLUT1 and VGAT (Figure 3E–L). These phenotypes were specific as plasmid-based reexpression of AP-2μ in AP-2μ KO neurons rescued defective endocytosis of these SEP-tagged SV proteins (Figure 3). Figure 3 with 1 supplement see all Download asset Open asset Clathrin-independent endocytic retrieval of select synaptic vesicle (SV) cargos by the clathrin adaptor adaptor protein complex 2 (AP-2) at physiological temperature. (A–D) Poststimulus retrieval of Syt1 and SV2A in the absence of AP-2 persists unaffected in response to 200 APs applied at 40 Hz. Average normalized traces of WT and AP-2μ KO derived neurons cotransfected with Syt1-SEP (A) or SV2A-SEP (C) and mRFP or rescued by reexpression of untagged AP-2µ subunit together with soluble mRFP (AP-2μ) to identify transfected neurons in response to 200 APs applied at 40 Hz. Quantification of the endocytic decay time constant (τ) of neurons expressing Syt1-SEP (B) (τWT = 17.17 ± 1.35 s, τAP-2μ KO = 21.53 ± 3.43 s, τAP-2μ KO+AP-2μ = 21.51 ± 3.91 s) or SV2A-SEP (D) (τWT = 18.33 ± 0.99 s, τAP-2μ KO = 20.45 ± 1.56 s, τAP-2μ KO+AP-2μ = 18.04 ± 1.16 s). Data shown represent the mean ± standard error of the mean (SEM): Syt1 (nWT = 7 images, nAP-2μ KO = 7 images, nAP-2μ KO+AP-2μ = 4 images; p(WT vs AP-2μ KO) = 0.4994; p(AP-2μ KO vs AP-2μ KO+AP2-μ) > 0.9999); SV2A (nWT = 44 images, nAP-2μ KO = 23 images, nAP-2μ KO+AP-2μ = 37 images; p(WT vs AP-2μ KO) = 0.4665; p(AP-2μ KO vs AP-2μ KO+AP-2μ) = 0.3943). One-way analysis of variance (ANOVA) with Tukey’s post-test. (E–L) Loss of AP-2 significantly delay the endocytic retrieval of other major SV proteins. Average normalized traces of WT and AP-2μ KO neurons cotransfected with VGLUT1-SEP (E), VGAT-SEP (G), Syp-SEP (I), Syb2-SEP (K), and mRFP or AP-2μ to rescue AP-2μ expression stimulated with 200 APs at 40 Hz. Endocytic decay time constants (τ) were calculated from WT, AP-2μ KO neurons, and AP-2µ KO neurons rescued by reexpression of AP-2µ expressing VGLUT1-SEP (F) (τWT = 13.52 ± 0.90 s, τAP-2μ KO = 30.33 ± 2.01 s, τAP-2μ KO+AP-2μ = 16.38 ± 1.00 s), VGAT-SEP (H) (τWT = 16.76 ± 0.87 s, τAP-2μ KO = 40.56 ± 9.21 s, τAP-2μ KO+AP-2μ = 21.77 ± 2.15 s), Syp-SEP (J) (τWT = 13.05 ± 0.74 s, τAP-2μ KO = 23.76 ± 1.72 s, τAP-2μ KO+AP-2μ = 14.89 ± 1.04 s), VGAT-SEP (D) (τWT = 18.33 ± 0.99 s, τAP-2μ KO = 20.45 ± 1.56 s, τAP-2μ KO+AP-2μ = 18.04 ± 1.16 s), and Syb2-SEP (L) (τWT = 20.27 ± 1.48 s, τAP-2μ KO = 46.34 ± 9.43 s, τAP-2μ KO+AP-2μ = 22.46 ± 1.21 s). Data shown represent the mean ± SEM: VGLUT1 (nWT = 37 images, nAP-2μ KO = 24 images, nAP-2μ KO+AP-2μ = 23 images; ****p(WT vs AP-2μ KO) < 0.0001; ****p(AP-2μ KO vs AP-2μ KO+AP-2μ) < 0.0001); VGAT (nWT = 34 images, nAP-2μ KO = 11 images, nAP-2μ KO+AP-2μ = 32 images; ****p(WT vs AP-2μ KO) < 0.0001; ***p(AP-2μ KO vs AP-2μ KO+AP-2μ) = 0.0008); Syp (nWT = 33 images, nAP-2μ KO = 29 images, nAP-2μ KO+AP-2μ = 37 images; ****p(WT vs AP-2μ KO) < 0.0001; ****p(AP-2μ KO vs AP-2μ KO+AP-2μ) < 0.0001); Syb2 (nWT = 15 images, nAP-2μ KO = 20 images, nAP-2μ KO+AP-2μ = 26 images; **p(WT vs AP-2μ KO) = 0.0083; **p(AP-2μ KO vs AP-2μ KO+AP-2μ) = 0.0052). One-way ANOVA with Tukey’s post-test. (M–P) Delayed poststimulus retrieval of major SV proteins in the absence of AP-2 is not caused by defects in re-acidification of endocytosed vesicles. Representative normalized traces of WT and AP-2μ KO neurons expressing VGLUT1-SEP (M) or SV2A-SEP (O) stimulated with 200 APs applied at 40 Hz and subjected to low pH imaging buffer before and after train stimulation. Fluorescence quenching by application of acidic buffer poststimulus (ΔF2) vs prestimulus (ΔF1) of VGLUT1-SEP (N) (nWT = 10 images, nAP-2µ KO = 6 images) or SV2A-SEP (P) (nWT = 8 images, nAP-2µ KO = 13 images) is taken as a measure to probe the SEP surface pool in WT and AP-2μ KO hippocampal neurons. Values for WT were set to 1. Data shown represent the mean ± SEM. VGLUT1: WT = 1.0 ± 0.1; AP-2µ KO = 1.8 ± 0.2. p = 0.0009. SV2: WT = 1.0 ± 0.1; AP-2µ KO = 1.1 ± 0.1. p = 0.4267. Two-sided unpaired t-test. Raw data can be found in Figure 3—source data 1. Figure 3—source data 1 Source data for Figure 3A-P. https://cdn.elifesciences.org/articles/71198/elife-71198-fig3-data1-v1.xlsx Download elife-71198-fig3-data1-v1.xlsx As elevated pHluorin signals could conceivably arise from defects in endocytosis or vesicle reacidification, we used a quench protocol in which acidic buffer is applied before and after neuronal stimulation with 200 APs to probe the accessibility of VGLUT1- or SV2A-SEP to externally applied acid (Figure 3M–P). These experiments showed that exocytosed VGLUT1-SEP accumulates on the surface of AP-2μ KO neurons (Figure 3M) as quantitatively evidenced by an increased ΔF2/ΔF1 ratio (Figure 3N). No difference in the fraction of surface-accumulated SV2A-SEP molecules was observed in AP-2μ KO compared to WT neurons (Figure 3O, P). Defective endocytosis of VGLUT1-SEP in the absence of AP-2 was further confirmed by probing the acid-resistant pool of endocytosed VGLUT1-SEP at 30 s poststimulation with 200 APs in the presence of folimycin, a selective inhibitor of vesicle reacidification by the V-ATPase (Figure 3—figure supplement 1C-F). These experiments demonstrate that AP-2 is required for VGLUT1-SEP endocytosis but is dispensable for vesicle reacidification. We challenged these data acquired with strong 200 AP stimulation by monitoring SV endocytosis in response to a milder stimulation paradigm that results in the exocytic fusion of the readily releasable pool of SVs (Hua et al., 2011; Murthy and Stevens, 1999), that is 50 APs applied at 20 Hz. While endocytosis of SV2A- and VGLUT1-SEP proceeded unperturbed in hippocampal neurons depleted of clathrin (Figure 4A–D), a substantial delay in the endocytosis of VGLUT1- but not SV2-SEP was observed in neurons lacking AP-2 (Figure 4E–H). No difference was found in the fraction of boutons responding to stimulation with 50 APs between WT and AP-2μ KO neurons (Figure 4—figure supplement 1A,B). At lower stimulation intensities (i.e., 10 or 20 APs), AP-2μ KO neurons displayed significantly attenuated exocytic responses (Figure 4—figure supplement 1B), possibly reflecting a reduced release probability originating from defects in SV reformation, akin to the reported phenotype of clathrin loss in hippocampal neurons (Watanabe et al., 2013; Watanabe et al., 2014). Figure 4 with 1 supplement see all Download asset Open asset Clathrin-independent endocytic retrieval of synaptic vesicle (SV) proteins mediated by adaptor protein complex 2 (AP-2) is independent of the stimulation strength at physiological temperature. (A–D) Lack of clathrin does not alter the endocytosis of SV2 and VGLUT1 in response to stimulation with 50 APs (i.e., a stimulus that releases the RRP) at physiological temperature. Average normalized traces of neurons transduced with lentivirus expressing nonspecific shRNA (shCTRL) or shRNA-targeting CHC (shCHC) and cotransfected with either SEP-tagged SV2A (A) or VGLUT1 (C) stimulated with 50 APs applied at 20 Hz at physiological temperature. Quantification of the endocytic decay time constant (τ) in neurons coexpressing SV2A-SEP (B) and shCTRL (20.97 ± 2.99 s) or shCHC (19.18 ± 2.22 s); and VGLUT1-SEP (D) and shCTRL (12.51 ± 1.62 s) or shCHC (13.68 ± 1.90 s). Data represent the mean ± standard error of the mean (SEM) for SV2A (nshCTRL = 14 images, nshCHC = 17 images; p = 0.6274) and for VGLUT1 (nshCTRL = 14 images, nshCHC = 15 images; p = 0.6468). Two-sided unpaired t-test. (E–H) Endocytosis delay for VGLUT1 but not for SV2A in neurons depleted of AP-2 when stimulated with a mild train of 50 APs. Average normalized traces of neurons from WT and AP-2μ KO mice transfected with either SV2A-SEP (E) or VGLUT1-SEP (G) in response of 50 APs applied at 20 Hz at physiological temperature. Quantification of the endocytic decay time constant (τ) of SV2A-SEP-expressing neurons (F) (τWT = 12.40 ± 1.05 s, τAP-2μ KO = 13.34 ± 0.95 s) or VGLUT1-SEP (H) (τWT = 11.61 ± 0.65 s, τAP-2μ KO = 23.96 ± 1.87 s). Data represent the mean ± SEM for SV2A (nWT = 13 images, nAP-2μ KO = 24 images; p = 0.5355) and for VGLUT1 (nWT = 21 images, nAP-2μ KO = 24 images; ****p < 0.0001). Two-sided unpaired t-test. Raw data can be found in Figure 4—source data 1. Figure 4—source data 1 Source data for Figure 4A-H. https://cdn.elifesciences.org/articles/71198/elife-71198-fig4-data1-v1.xlsx Download elife-71198-fig4-data1-v1.xlsx Collectively, these data unravel a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos including VGLUT1 and VGAT at physiological temperature, while endocytosis of Syt1 or SV2A proceeds with unaltered kinetics in the absence of AP-2. CIE of endogenous VGAT depends on the clathrin adaptor AP-2 As optical imaging of SEP reporters may lead to artifacts caused by overexpression of exogenous SV proteins (Opazo et al., 2010), we analyzed the internalization kinetics of endogenous VGAT using antibodies directed against its luminal domain coupled to the pH-sensitive fluorophore CypHer5E. The cyanine-based dye CypHer5E is quenched at neutral pH but exhibits bright fluorescence when present in the acidic lumen of SVs (Hua et al., 2011) and, thus can serve as a tracer for the recycling of endogenous SV proteins when it is preloaded into SVs (e.g., by high-frequency stimulation or spontaneous labeling, see Materials and methods) prior to the measurements (Figure 5A). First, we probed the effects of AP-2μ KO on VGAT endocytosis. Loss of AP-2 severely delayed the endocytic retrieval of endogenous VGAT in response to train stimulation with either 200 APs (Figure 5B, C) or 50 APs (Figure 5D, E) at physiological temperature, consistent with our results from exogenously expressed VGAT-SEP (see Figure 3). To determine whether the requirement for AP-2 reflects a function for CME in the retrieval of endogenous VGAT, we examined the effects of genetic or pharmacological blockade of clathrin function. Lentiviral shRNA-mediated depletion of clathrin had no effect on the endocytic retrieval of endogenous VGAT in response to either strong (e.g., train of 200 APs applied at 40 Hz) (Figure 5F, G) or mild stimulation (50 APs at 20 Hz) (Figure 5H, I) at physiological temperature. Similar results were obtained, if clathrin function was perturbed pharmacologically by acute inhibition in the presence of Pitstop2 in either WT or clathrin-depleted neurons (F