Abstract
Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila. Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.
Significance Statement
Understanding the regulation of synaptic development and refinement could provide mechanistic insights into the neural basis of fundamental processes such as learning, memory and adaptation. In this study, we describe the role of σ2-adaptin in attenuating neuromuscular junction (NMJ) growth signaling and show its involvement in the trafficking of Thickveins receptor at the Drosophila NMJ. σ2-Adaptin interacts genetically with the BMP pathway components and NMJ synapses lacking σ2-adaptin show reduced levels of the endosomal recycling marker, Rab11. Our study contributes to a broader understanding of AP2 complex-dependent regulation of synaptic growth signaling, which might have implications in neurodevelopment under conditions where endocytosis and trafficking of the BMP receptors are perturbed.
Introduction
Synapse development and refinement is an interplay of multiple signaling pathways, namely, the Bone morphogenetic protein (BMP), Wingless (Wnt), and Ubiquitin-Proteasome-mediated protein degradation (Highwire-Wallenda) pathways (McCabe et al., 2003, 2004; Franco et al., 2004; Collins et al., 2006; Ball et al., 2010; Budnik and Salinas, 2011; Piccioli and Littleton, 2014; Choudhury et al., 2016). Besides, there are also pathways mediated by endocytic, cytoskeletal, and actin regulatory proteins (Franco et al., 2004; Piccioli and Littleton, 2014; Choudhury et al., 2016). Understanding the crosstalk among them is crucial to our understanding of the processes that regulate synapse development, refinement, and plasticity (Deshpande and Rodal, 2016). BMP signaling pathway at the Drosophila neuromuscular junction (NMJ) synapses is a well-studied growth-promoting pathway (Marqués et al., 2002; McCabe et al., 2003; O’Connor-Giles et al., 2008; Ball et al., 2010). The canonical BMP signaling is dependent on phosphorylated Smad (pMad in Drosophila) and its translocation into the motor neuron nuclei, followed by the transcription of BMP target genes (Keshishian and Kim, 2004; O’Connor-Giles et al., 2008; Ball et al., 2010; Kim and Marqués, 2012; Sulkowski et al., 2016; Vuilleumier et al., 2019).
At the Drosophila NMJ, the retrograde BMP signaling is initiated by secretion of Glass bottom boat (Gbb) from the muscle and the motor neurons (Goold and Davis, 2007). Gbb binds to Wit (a Type II receptor) as well as Tkv and Sax (Type I receptors) at the presynaptic nerve terminals to control NMJ growth and function (Aberle et al., 2002; Marqués et al., 2002; McCabe et al., 2003). Gbb binding to Wit triggers the tetramerization of BMP receptors that, in turn, phosphorylates the Smad transcription factor, Mothers against decapentaplegic (Mad). Following BMP activation, these receptors are endocytosed for retrograde transport to the motor neuron nuclei in the soma (Rodal et al., 2011; Smith et al., 2012; Vanlandingham et al., 2013).
Multiple studies have shown a tight correlation between defective endocytosis, altered synapse growth, and elevated synaptic pMad levels, implicating increased BMP signaling in synaptic development (O’Connor-Giles et al., 2008; Ball et al., 2010; Nahm et al., 2010; Shi et al., 2013; Piccioli and Littleton, 2014). One such study has shown that Nwk, an F-BAR and SH3 domain-containing protein that negatively regulates synaptic growth, interacts with Tkv along with Dap160 and Dynamin (both endocytic proteins) to attenuate retrograde BMP signaling during NMJ growth (O’Connor-Giles et al., 2008). Endocytic and endosomal pathways are, therefore, critical to controlling both the activity and localization of signaling proteins that regulate synaptic growth (Rodal et al., 2011; Cosker and Segal, 2014). Defects in intracellular trafficking can also lead to enhanced signaling from the cellular compartments (like endosomes), affecting the synapse development (Di Fiore and De Camilli, 2001; Dubois et al., 2001; Sweeney and Davis, 2002; Rodal et al., 2011). In the neuronal context, the efficacy of intercellular signaling is regulated by the trafficking of activated receptor/ligand complexes following endocytosis from the presynaptic membrane.
Tightly regulated endocytic transport of BMP receptors relies on the spatiotemporal regulation of Rab GTPase function (Kelly et al., 2012). The Rab-family of GTPases regulates the progression of receptor endocytosis and participates in the successive steps of membrane maturation, receptor transport, and turnover (Horgan and McCaffrey, 2011). In particular, Rab5 regulates vesicle formation and is associated with early endosomes, while Rab7 and Rab11 associate with late and recycling endosomes, respectively (Chavrier et al., 1990; Ullrich et al., 1996). Endosomal trafficking of BMP signaling complexes at the nerve terminals is known to fine-tune the intensity and persistence of BMP signaling (Cosker and Segal, 2014; Deshpande and Rodal, 2016). Altered distribution or misregulation of Rab11 has been shown to suppress Tkv trafficking from early endosome to presynaptic membrane resulting in elevated BMP signaling (O’Connor-Giles et al., 2008; Rodal et al., 2008; Liu et al., 2014; Deshpande and Rodal, 2016).
An important, yet enigmatic question, is the correlation between defective Clathrin-mediated endocytosis (CME) and aberrant synaptic growth. For instance, it is not known whether specific modes of endocytosis externalize specific cargos whose trafficking defect perturbs synaptic signaling. CME, the major endocytic pathway, is required not only for basal synaptic transmission at nerve terminals but also for synapse development (Koh et al., 2004; Dickman et al., 2006; Choudhury et al., 2016). For instance, perturbations in CME resulting from mutations in Dynamin, AP2 subunits, Endo, or Synj all exhibit NMJ structural defects resulting in increased number but decreased size of synaptic boutons in Drosophila (Dickman et al., 2006; Choudhury et al., 2016). It is unclear whether the NMJ structural defects associated with the endocytic mutants is a consequence of deficient endosomal trafficking leading to aberrant synaptic signaling. It is likely that perturbing CME deregulates signaling modules of BMP pathway that leads to elevated pMad in the endocytic mutants (O’Connor-Giles et al., 2008; Choudhury et al., 2016).
In central synapses, AP2-dependent CME is dispensable for membrane regeneration from the presynaptic plasma membrane following high-frequency nerve stimulation (Kononenko et al., 2014). However, the critical role of CME in generating vesicles from endosome-like structures following bulk membrane endocytosis cannot be ruled out (Watanabe et al., 2014). Previous studies support a model in which compromised CME can lead to defective signalosome trafficking by trapping signaling molecules in endosomes or intermediate structures of the endosomal pathway (Dubois et al., 2001; Lloyd et al., 2002; Sweeney and Davis, 2002; Wang et al., 2007; Papagiannouli et al., 2019; Joseph et al., 2020). Another study has shown clathrin-independent role of the AP2 in the endocytic retrieval of select synaptic vesicle (SV) cargos from the presynaptic cell surface (López-Hernández et al., 2021).
Our previous study has shown elevated levels of synaptic as well as motor-nuclei pMad in σ2-adaptin mutants (Choudhury et al., 2016). In order to investigate the underlying signaling mechanisms leading to elevated pMad levels, we performed epistatic interactions between σ2-adaptin mutants with the components of BMP signaling. Introducing a mutant copy of tkv in σ2-adaptin mutants significantly reduces the NMJ overgrowth. Conversely, introducing a mutant copy of inhibitory Smad, Dad in a heterozygous σ2-adaptin background leads to NMJ overgrowth. Ultrastructural analysis of NMJ revealed accumulation of large vesicles and supports a role of σ2-adaptin in generating signalosomes containing vesicles, possibly from endosomal structures. Further analysis of vesicular trafficking using endosomal markers shows that Rab11 is reduced in σ2-adaptin mutant NMJ synapses. Thus, our studies reveal a novel function of σ2-adaptin in attenuating BMP signaling by facilitating trafficking and recycling of the Tkv receptor.
Materials and Methods
Fly stock
Flies were grown and maintained at 25°C temperature in a standard cornmeal medium as described earlier (Choudhury et al., 2016). Both males and females were used for experiments in the current study. w1118 was used as control unless otherwise stated. Two σ2-adaptin alleles were used: AP2σang7,which is a hypomorphic allele obtained through a P-element mobilization of SyndEP877 located 2416 bp upstream of the Syndapin ORF, and AP2σKG02457, which is a P-element insertion in the third exon of the σ2-adaptin ORF making it a null allele (Choudhury et al., 2016). All the mutants, controls, and rescued larvae of either sex were grown under noncrowded conditions on apple agar plates with yeast paste dollop. The following stocks were obtained from Bloomington Drosophila Stock Center (BDSC): tkv7 (BL3242), Med13 (BL7340), witA12 (BL5173), Dadj1E4 (BL10305), AP2σKG02457 (BL13478), UAS-Rab11-GFP (BL8506), Madk00237 (BL10474), α-adaptin dsRNA (BL32866), Clc dsRNA (BL27496), Synj1 (BL24883), Synj2 (BL24884). Other lines used in this study are: D42-Gal4 (Yeh et al., 1995; Sanyal, 2009), UAS-tkv-EGFP (BL51653; Ayyaz et al., 2015), UAS-YFP-Rab11S25N (BL9792; Guan et al., 2020), UAS-Rab11Q70L-GFP (BL23260; Sorvina et al., 2016), Rab11ex2 and Rab1193Bi (Khodosh et al., 2006).
Antibodies and immunocytochemistry
Wandering third instar larvae were dissected in cold calcium-free HL3 saline (70 mm NaCl, 5 mm KCl, 20 mm MgCl2, 10 mm NaHCO3, 5 mm Trehalose, 115 mm sucrose, and 5 mm HEPES, pH 7.2) to expose the NMJs and fixed in 4% paraformaldehyde in PBS (pH 7.2) for 30 min at room temperature. Fillets were then washed in PBS containing 0.15% Triton X-100, blocked for 1 h with 5% bovine serum albumin (BSA), followed by overnight incubation with primary antibody at 4°C. The monoclonal antibody anti-CSP (1:100) was obtained from the Developmental Studies Hybridoma Bank (DSHB). The polyclonal antibody against Rab5 (1:500; Mottola et al., 2010) was a gift from Marino Zerial, Max Planck Institute, Germany. The polyclonal Rab7 (1:500) and Rab11 (1:500; Tanaka and Nakamura, 2008; West et al., 2015) antibodies were a gift from Tsubasa Tanaka, RIKEN Center for Developmental Biology, Japan. The pMad antibody was a gift from Peter ten Dijke and used at 1:1000 dilution (Persson et al., 1998). The secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 568 (Invitrogen, Thermo Fisher Scientific) were used at 1:800 dilution. The Alexa Fluor 488 or Rhodamine-conjugated anti-HRP (Jackson ImmunoResearch) were used at 1:800 dilution. Stained larval fillets were mounted in VECTASHIELD (Vector Laboratories). All the images were captured with a laser scanning confocal microscope (LSM780, Carl Zeiss or FV3000, Olympus Corporation).
Electrophysiology
All intracellular recordings were performed on wandering third instar larvae as described previously (Choudhury et al., 2016). Briefly, HL3 buffer containing 1.5 mm Ca2+ was used for larval dissection. Recordings from muscle 6 of A2 hemisegment were performed using sharp glass electrodes having a resistance of 20–25 MΩ. Miniature excitatory junction potentials (mEJPs) were recorded for 60 s, followed by recordings of EJPs at 1 Hz stimulation. For High-frequency recording, nerves were stimulated at 10 Hz, and EJPs were recorded for 5 min. For recording EJPs, stimulation pulse was delivered using Grass S88 stimulator (Grass Instruments, Astro-Med, Inc). The signals were amplified using Axoclamp 900A, digitized using Digidata 1440A, and acquired using pClamp10 software (Molecular Devices). Muscles with resting membrane potential between −60 and −75 mV were used for analysis. The data were analyzed using the Mini Analysis program (Synaptosoft, Decatur).
Intensity profile
Confocal images of muscle four NMJ at A2 hemisegment were used to plot the intensity profile. A single bouton section was used, and the intensities of Tkv and HRP were analyzed by drawing a line across the bouton using Fiji/ImageJ software. The graph was plotted in Microsoft Excel using the intensity values obtained from Fiji/ImageJ software. As the intensity of Tkv in control was too low to plot the graph, all the intensity values were multiplied two-fold.
Electron microscopy
TEM was performed as described previously (Deivasigamani et al., 2015). Briefly, third instar larvae were dissected in cold PBS. The larval fillets were then fixed in 0.12 m cacodylate buffer containing 2% glutaraldehyde for 10 min at room temperature, transferred to a fresh fixative, and kept overnight at 4°C. The fillets were postfixed for 1 h with 2% osmium tetroxide (OsO4) solution prepared in 0.12 m cacodylate buffer. The samples were rinsed with 0.12 m cacodylate buffer followed by washes with distilled water to avoid precipitation of cacodylate with Uranyl acetate. Subsequently, the samples were subjected to en bloc staining with 2% uranyl acetate. The stained fillets were again washed with distilled water and dehydrated using graded solutions of ethanol before final infiltration of the samples through propylene oxide for 30 min. Stained and dehydrated fillets were embedded in epoxy resin and hardened overnight at 60°C. Muscles embedded in epoxy resin were sectioned at 60 nm. Ultrathin sections of the muscles stained with 2% uranyl acetate (in 70% ethanol) and 1% aqueous lead citrate were examined at 120 KV on a Tecnai G2 Spirit BioTWIN (FEI) electron microscope. The number of synaptic vesicles per bouton were counted manually using the Multi-point tool in ImageJ/Fiji software and then divided by their respective bouton areas to obtain the vesicle density/μm2 area of a bouton. For vesicle size, diameters of at least 100 vesicles from 10 bouton sections of each genotype were used for quantification. For calculating the number of synaptic vesicles docked at the active zones, only those vesicles that were touching the T-bar platform were counted. For calculating the SSR thickness, the scale bar in the images was first calibrated to the number of pixels using the Set scale function in ImageJ/Fiji software. This was followed by using the Straight-line tool to draw a line across the SSR and Measure tool to calculate the thickness.
Western blot analysis
The western blot analysis was done as previously described previouusly (Choudhury et al., 2016). Briefly, VNC from wandering third instar larvae were dissected out in ice-cold HL3 buffer and homogenized in buffer containing 50 mm Tris-HCl, pH 6.8, 25 mm KCl, 2 mm EDTA, 0.3 m sucrose, and 2% SDS in water. The homogenized sample was then mixed with an equal volume of 2× Laemmli buffer. The protein equivalent to 50 μg was separated on 12% SDS-PAGE and transferred to Hybond-LFP PVDF membrane (GE Healthcare, GE Healthcare Life Sciences). The membrane was then blocked with 5% skimmed milk for 1 h, followed by overnight incubation with anti-Rab11 (1:2000) and anti-Tubulin (1:5000) antibody. IRDye 800 (1:10 000) was used as a secondary antibody, and signals were visualized on LI-COR Odyssey platform. The density of Western bands was quantified using ImageJ/Fiji software.
Quantification and statistical analysis
For fluorescence quantification, images were captured using a laser scanning confocal microscope (LSM780; Carl Zeiss or FV3000, Olympus). All the control and experimental fillets were processed in the same way, and the fluorescence images were captured under the same settings for every experimental set. For bouton quantification, CSP-labeled structures were counted at muscle 6/7 of A2 hemisegment. The number of boutons from each NMJ was normalized to the respective muscle area. To calculate the bouton number, NMJs from A2 hemisegment were captured using a plan apochromat 40× objective, 1.4 NA and all the CSP positive boutons were counted manually in ImageJ/Fiji software. For muscle area quantification, images from A2 hemisegment were captured using 20× objective, and the area was quantified using ZEN2 software (Carl Zeiss, Germany). For bouton number quantification, the total number of boutons per NMJ was divided by the respective muscle areas. For fluorescence intensity quantification, NMJs from muscle four were captured using a plan apochromat 60×, 1.4 NA objective. For each NMJ, the fluorescence intensity from each bouton was subtracted from the background intensity, and the average intensity was normalized to the control. The fluorescence intensity was calculated using ImageJ/Fiji software. For bouton area quantification, NMJs from muscle 6/7 at A2 hemisegment were captured, and the area was calculated by drawing a free-hand sketch around CSP positive bouton using ImageJ/Fiji software. For multiple comparisons, one-way ANOVA followed by post hoc Tukey’s test, or Student’s t test was used. GraphPad Prism 8 was used to plot the graph. Error bars in all the histograms represent SEM. *p < 0.5, **p < 0.01, ***p < 0.001.
Results
σ2-adaptin genetically interacts with regulators of BMP signaling
In a previous study, we showed σ2-adaptin mutants caused NMJ overgrowth and upregulation of pMad, an effector of the BMP pathway. Two previously characterized σ2-adaptin alleles, AP2σang7, a strong hypomorph and AP2σKG02457, a null allele was used in the current study (Choudhury et al., 2016). To explore the role σ2-adaptin in regulating BMP signaling at the NMJ, we first assessed the epistatic interaction between σ2-adaptin and components of the BMP-signaling pathway. We found that introducing one mutant copy of tkv, Med, or Mad in σ2-adaptin mutant background significantly suppressed the synaptic overgrowth phenotype in these animals (Fig. 1A–F). The number of boutons normalized to the muscle area was significantly rescued in tkv7/+; AP2σKG02457/AP2σang7 (1.91 ± 0.12, p ≤ 0.001), Med13, AP2σKG02457/AP2σang7 (1.82 ± 0.07, p ≤ 0.001), and Madk00237/+; AP2σKG02457/AP2σang7 (2.25 ± 0.11, p ≤ 0.001) when compared with AP2σKG02457/AP2σang7 (2.85 ± 0.08, p ≤ 0.001). However, there was no significant difference between w1118 control, heterozygous AP2σKG02457/+, and tkv7/+ (w1118: 1.26 ± 0.06, AP2σKG02457/+: 1.33 ± 0.03 and tkv7/+: 1.34 ± 0.05; Fig. 1G).
Downregulating BMP signaling components reduced the synaptic overgrowth and clustering in the σ2-adaptin mutant. A–F′, Confocal images of NMJ synapses at muscles 6/7 of A2 hemisegment showing synaptic growth in (A, A′) w1118 (Control), (B, B′) tkv7/+, (C, C′) AP2σKG02457/AP2σang7, (D, D′) tkv7/+; AP2σKG02457/AP2σang7, (E, E′) Med13, AP2σKG02457/AP2σang7, and (F, F′) Madk00237/+; AP2σKG02457/AP2σang7 double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Reducing the levels of BMP signaling components in the AP2σKG02457/AP2σang7 background reduces the synaptic overgrowth. Scale bar in F (for A–F) and F′ (for A′–F′) represents 20 and 5 μm, respectively. G, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.26 ± 0.06), AP2σKG02457/+ (1.33 ± 0.03), tkv7/+ (1.34 ± 0.05), AP2σKG02457/AP2σang7 (2.85 ± 0.08), tkv7/+; AP2σKG02457/AP2σang7 (1.91 ± 0.12), Med13, AP2σKG02457/AP2σang7 (1.82 ± 0.07), and Madk00237/+; AP2σKG02457/AP2σang7 (2.25 ± 0.11). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. H, Histogram showing the average bouton area from muscle 6/7 NMJ at A2 hemisegment in control animals (9.57 ± 0.53), AP2σKG02457/+ (8.89 ± 0.35), tkv7/+ (9.11 ± 0.46), AP2σKG02457/AP2σang7 (1.98 ± 0.03), tkv7/+; AP2σKG02457/AP2σang7 (2.85 ± 0.05), Med13, AP2σKG02457/AP2σang7 (2.79 ± 0.05), and Madk00237/+; AP2σKG02457/AP2σang7 (3.86 ± 0.14). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. I, Histogram showing the average NMJ length normalized with muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (3.43 ± 0.18), AP2σKG02457/+ (2.5 ± 0.22), tkv7/+ (2.5 ± 0.14), AP2σKG02457/AP2σang7 (3.49 ± 0.15), tkv7/+; AP2σKG02457/AP2σang7 (3.91 ± 0.39), Med13, AP2σKG02457/AP2σang7 (3.95 ± 0.25), and Madk00237/+; AP2σKG02457/AP2σang7 (4.08 ± 0.22). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. J, Histogram showing the average number of branches from muscle 6/7 NMJ at A2 hemisegment in control animals (9.56 ± 0.71), AP2σKG02457/+ (8.77 ± 0.43), tkv7/+ (9.11 ± 0.53), AP2σKG02457/AP2σang7 (7.8 ± 0.36), tkv7/+; AP2σKG02457/AP2σang7 (8.88 ± 0.55), Med13, AP2σKG02457/AP2σang7 (9.25 ± 0.56), and Madk00237/+; AP2σKG02457/AP2σang7 (10 ± 0.5). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. Data supporting the rescue of σ2-adaptin mutant phenotypes by reducing Wit receptors is shown in Extended Data Figure 1-1.
Extended Data Figure 1-1
Reducing levels of Wit receptor rescues the bouton number in σ2-adaptin mutant. A–C, Confocal images of NMJ synapses at muscles 6/7 NMJ at A2 hemisegment showing the synaptic growth in control (A), AP2σKG02457/AP2σang7 (B), witA12, AP2σKG02457/AP2σang7 (C), double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Reducing the levels of BMP Type II receptor in the AP2σKG02457/AP2σang7 background reduces the synaptic overgrowth. Scale bar in C represents 10 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.33 ± 0.03), AP2σKG02457/AP2σang7 (2.50 ± 0.11), and witA12, AP2σKG02457/AP2σang7 (2.0 ± 0.07). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01, ***p < 0.001. Download Figure 1-1, TIF file.
Consistent with the above observations, we found that mutating one copy of wit significantly reduced the σ2-adaptin induced synaptic overgrowth (witA12, AP2σKG02457/AP2σang7: 2.00 ± 0.08 vs AP2σKG02457/AP2σang7: 2.50 ± 0.11; p ≤ 0.01; Extended Data Fig. 1-1). Since elevated BMP signaling results in the formation of smaller boutons, we quantified the bouton area in these genotypes. We found that introducing one copy of tkv7 (tkv7/+; AP2σKG02457/AP2σang7: 2.9 ± 0.05, p ≤ 0.01), Med13 (Med13, AP2σKG02457/AP2σang7: 2.79 ± 0.05, p ≤ 0.05), and Madk00237/+; AP2σKG02457/AP2σang7 (3.86 ± 0.15, p ≤ 0.001) in σ2-adaptin mutant background partially restored the bouton area when compared with σ2-adaptin mutant alone (AP2σKG02457/AP2σang7:1.98 ± 0.03; w1118 controls: 9.5 ± 0.53; Fig. 1A′–F′,H). Compared with w1118 controls, no significant difference was found in the branching and length of the NMJ in these genotypes (Fig. 1I,J). Thus, our data suggest that σ2-adaptin genetically interacts with the BMP receptors to regulate NMJ bouton number and area.
To further strengthen our observation that σ2-adaptin attenuates BMP signaling, we tested the interaction between σ2-adaptin and Dad, a negative regulator of BMP signaling (O’Connor-Giles et al., 2008; L Zhao et al., 2013). We first examined the total number of synaptic boutons in transheterozygotes of σ2-adaptin and Dad mutants (Fig. 2). While the number of boutons in larvae heterozygous for σ2-adaptin [AP2σKG02457/+ (1.23 ± 0.05)] and Dad [Dadj1E4/+ (1.39 ± 0.09)] was comparable with w1118 control (1.26 ± 0.06), transheterozygous Dadj1E4/AP2σKG02457 (1.89 ± 0.09, p ≤ 0.001) showed significantly higher bouton number when compared with w1118 controls (Fig. 2H). However, there was no significant difference in the bouton area in transheterozygotes of σ2-adaptin and Dad mutants Dadj1E4/AP2σKG02457 (8.51 ± 0.61) when compared with w1118 control (10.91 ± 0.87; Fig. 2I).
σ2-adaptin genetically interacts with Dad, the inhibitory Smad of BMP signaling. A–G, Confocal images of NMJ synapses at muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in (A) Control, (B) AP2σKG02457/+, (C) Dadj1E4/+, (D) AP2σKG02457/Dadj1E4, (E) UAS-Dad/+; D42-Gal4/+, (F) AP2σKG02457/AP2σang7, and (G) UAS-Dad/+; D42-Gal4, AP2σang7/AP2σKG02457 double immunolabeled with a presynaptic synaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Note that trans-heterozygotes of σ2-adaptin and Dad mutants show significantly increased synaptic growth. Scale bar in G represents 20 μm. H, Histogram showing average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.26 ± 0.06), AP2σKG02457/+ (1.23 ± 0.05), Dadj1E4/+ (1.39 ± 0.09), AP2σKG02457/Dadj1E4 (1.89 ± 0.09), UAS-Dad/+; D42-Gal4/+ (1.1 ± 0.07), AP2σKG02457/AP2σang7 (3.1 ± 0.18), and UAS-Dad/+; D42-Gal4, AP2σang7/AP2σKG02457 (1.87 ± 0.15). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001; ns, not significant. I, Histogram showing average bouton area from muscle 6/7 NMJ at A2 hemisegment in control animals (10.91 ± 0.87), AP2σKG02457/+ (9.6 ± 0.63), Dadj1E4/+ (11.16 ± 0.95), AP2σKG02457/Dadj1E4 (8.5 ± 0.61), UAS-Dad/+; D42-Gal4/+ (8.42 ± 0.36), AP2σKG02457/AP2σang7 (2.38 ± 0.11), and UAS-Dad/+; D42-Gal4, AP2σang7/AP2σKG02457 (5.94 ± 0.3). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. *p < 0.05, **p < 0.01; ns, not significant.
Next, we examined whether neuronally expressing UAS-Dad in σ2-adaptin mutant could rescue the synaptic overgrowth phenotype (Fig. 2E,F). Neuronal expression of Dad in σ2-adaptin mutant background UAS-Dad/+; D42-Gal4, AP2σang7/AP2σKG02457 (1.87 ± 0.15, p ≤ 0.001) significantly rescued the synaptic overgrowth in σ2-adaptin mutant AP2σang7/AP2σKG02457 (3.1 ± 0.18, p ≤ 0.001; Fig. 2G). Moreover, we found that neuronal expression of Dad in σ2-adaptin mutant background partially rescued the average bouton area (UAS-Dad/+; D42-Gal4, AP2σang7/AP2σKG02457: 5.94 ± 0.29, p ≤ 0.001; Fig. 2I). Taken together, these data suggest that σ2-adaptin regulates BMP signaling to attenuate synaptic growth at the Drosophila NMJ.
The functional and morphologic aspects of σ2-adaptin can be genetically discriminated
Since one mutant copy of BMP receptors in the σ2-adaptin mutant background significantly restores the morphologic defects, we asked whether the electrophysiological defects associated with the σ2-adaptin mutant could also be rescued. To address this, we measured evoked excitatory junction potential (EJP), quantal content (QC), and high-frequency intracellular recording on w1118 control, heterozygous tkv7/+, AP2σKG02457/AP2σang7, and tkv7/+; AP2σKG02457/AP2σang7 larvae (Fig. 3). We found that both mEJP amplitude and quantal content in tkv7/+; AP2σKG02457/AP2σang7 (mEJP amplitude = 1.01 ± 0.08, QC = 38.20 ± 3.09) were not significantly different from σ2-adaptin mutant AP2σKG02457/AP2σang7 (mEJP = 1.04 ± 0.09, QC = 39.29 ± 5.81; Fig. 3A–D). The scatter plot for mEJP amplitude distribution showed that both σ2-adaptin mutant and tkv7/+; AP2σKG02457/AP2σang7 had more mEJPs with higher amplitude compared with w1118 controls (Fig. 3C). Furthermore, we found that reducing the levels of Tkv receptor did not rescue the activity-dependent decline in EJP amplitude. The line plot showed that both σ2-adaptin mutant and tkv7/+; AP2σKG02457/AP2σang7 followed the same trend of EJP amplitude decline during the high frequency (10 Hz) stimulations (Fig. 3E,F). Moreover, there was no significant difference between w1118 and tkv7/+ controls in mEJP amplitude, QC, and high frequency recordings (w1118: mEJP = 0.65 ± 0.03, QC = 61.92 ± 4.02; tkv7/+: mEJP = 0.71 ± 0.05, QC = 69.77 ± 6.98; Fig. 3B,D,F). These data suggest that while reducing BMP signaling by lowering Tkv receptors in σ2-adaptin mutant significantly rescued neuronal overgrowth, it did not restore the physiological deficiencies in σ2-adaptin mutants indicating that functional and morphologic defects in σ2-adaptin mutants could be independent of one another.
Structural and functional deficits in σ2-adaptin mutant can be genetically discriminated. A, Representative traces of mEJP in control, tkv7/+, heteroallelic AP2σKG02457/AP2σang7, and tkv7/+; AP2σKG02457/AP2σang7 larvae. B, Quantification of average mEJP amplitude in control (0.65 ± 0.03), tkv7/+ (0.71 ± 0.05), heteroallelic AP2σKG02457/AP2σang7 (1.04 ± 0.09) and tkv7/+; AP2σKG02457/AP2σang7 (1.01 ± 0.08). At least eight NMJ recordings of each genotype were used for quantification. Error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. C, Scatter plot shows quantification of mEJP amplitude distribution in control, tkv7/+, heteroallelic AP2σKG02457/AP2σang7 and tkv7/+; AP2σKG02457/AP2σang7. Note that events with mEJPs greater than 4 mV occurred more frequently in AP2σKG02457/AP2σang7 and tkv7/+; AP2σKG02457/AP2σang7 compared with controls. D, Quantification of quantal content in control (61.92 ± 4.02), tkv7/+ (69.77 ± 6.98), AP2σKG02457/AP2σang7 (39.29 ± 5.81) and tkv7/+; AP2σKG02457/AP2σang7 (38.2 ± 3.09). At least eight NMJ recordings of each genotype were used for quantification. Error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Representative traces of EJPs under high-frequency stimulation of control, tkv7/+, heteroallelic AP2σKG02457/AP2σang7 and tkv7/+; AP2σKG02457/AP2σang7 larvae stimulated at 10 Hz for 5 min in 1.5 mm Ca2+ containing HL3. F, Line plot shows quantification of EJP amplitude during 10-Hz high-frequency recordings in control, tkv7/+, AP2σKG02457/AP2σang7, and tkv7/+; AP2σKG02457/AP2σang7. Note that the decline in EJP amplitude in σ2-adaptin mutant could not be rescued by reducing the levels of Tkv.
Loss of σ2-adaptin leads to the accumulation of endosome-like structures at the NMJ
Studies have shown that internalization and recycling of the membrane and BMP receptors depend on CME and endosomal proteins such as Rab5 and Rab11 (Mitchell et al., 2004; Liu et al., 2014). Mutants with defects in endocytosis and endosomal trafficking cause functional and ultrastructural abnormalities at the synapses. To get a deeper understanding of how loss of σ2-adaptin affects synapse ultrastructure, we performed transmission electron microscopy on σ2-adaptin mutant NMJs. Interestingly, ultrastructural analysis of σ2-adaptin deficient synapses showed an accumulation of large endosome-like structures, similar to what has been shown in mutants that affect endocytic and endosomal recycling machinery such as clathrin (Kasprowicz et al., 2008; Kawasaki et al., 2011), AP180 (B Zhang et al., 1998), Rab5 (Shimizu et al., 2003), Rab8 (West et al., 2015), and Rab11 (Inoshita et al., 2017). We found decreased synaptic vesicle (SV) density (w1118: 85.18 ± 12.26 vs AP2σKG02457/AP2σang7: 28.02 ± 14, p ≤ 0.01) and increased size of the SVs in the σ2-adaptin mutants (w1118: 43.16 ± 0.94 vs AP2σKG02457/AP2σang7: 71.53 ± 3.70, p ≤ 0.001). While there was no change in the number of SVs docked at the active zones (w1118: 0.91 ± 0.20 vs AP2σKG02457/AP2σang7: 0.78 ± 0.28, p > 0.05), we found significantly decreased thickness of the subsynaptic reticulum (SSR; w1118: 0.68 ± 0.03 vs AP2σKG02457/AP2σang7: 0.45 ± 0.03, p ≤ 0.001) in the σ2-adaptin mutants. These ultrastructural defects were rescued on ubiquitous expression of a σ2-adaptin transgene [actin5C-Gal4/+; AP2σKG02457, UAS-AP2σ/AP2σang7: SV density (107.9 ± 11.32), SV size (43.1 ± 1.46), and SSR thickness (0.61 ± 0.01); Fig. 4A–G]. Together, these data indicate that compromised regeneration of vesicles from the presynaptic membrane and defective membrane recycling in σ2-adaptin mutants results in the accumulation of large endosome-like structures.
σ2-adaptin mutant synapses show accumulation of large endosome-like vesicular structure. A–C, Electron micrographs of third instar Type Ib boutons of control (A), AP2σKG02457/AP2σang7 (B), and actin5C/+; AP2σKG02457, UAS-AP2σ/AP2σang7 (C). Arrows point to the large endosome-like vesicular structures observed in AP2σKG02457/AP2σang7 boutons but are absent in control and rescued boutons. The insets show a magnified area around the active zones. The presynaptic compartment is pseudocolored in cyan, and the subsynaptic reticulum is marked in green. Scale bar represents 500 nm. D, Histogram showing average vesicle diameter in control (43.16 ± 0.94), AP2σKG02457/AP2σang7 (71.53 ± 3.7), and actin5C/+; AP2σKG02457, UAS-AP2σ/AP2σang7 (43.1 ± 1.46). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001, **p < 0.01. E, Histogram showing SV density per unit area in control (85.18 ± 12.26), AP2σKG02457/AP2σang7 (28.02 ± 14), and actin5C/+; AP2σKG02457, UAS-AP2σ/AP2σang7 (107.9 ± 11.32). At least 10 images from three different larvae per genotype were used for quantification. Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001, **p < 0.01. F, Histogram showing average number of synaptic vesicles docked at the active zones in control (0.91 ± 0.20), AP2σKG02457/AP2σang7 (0.78 ± 0.28), and actin5C/+; AP2σKG02457, UAS-AP2σ/AP2σang7 (0.86 ± 0.34). At least five images from three different larvae per genotype were used for quantification. Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. G, Histogram showing SSR thickness in control (0.68 ± 0.03), AP2σKG02457/AP2σang7 (0.45 ± 0.03), and actin5C/+; AP2σKG02457, UAS-AP2σ/AP2σang7 (0.61 ± 0.01). At least five images from three different larvae per genotype were used for quantification. Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001.
σ2-adaptin mutants display increased Tkv receptors at the NMJ
AP2 complex has been shown to regulate CME and activity-dependent vesicle regeneration from endosome-like vacuoles (Kononenko et al., 2014; Kadlecova et al., 2017). Since the ultrastructural analysis of σ2-adaptin mutants revealed an accumulation of large endosome-like membranous structures similar to mutants with perturbed endocytosis or endosomal trafficking such as Rab5, Rab8, and Rab11 mutants (Shimizu et al., 2003; West et al., 2015; Inoshita et al., 2017), we hypothesized that σ2-adaptin could be involved either in endocytosis of BMP receptors from the presynaptic membrane or in the endosome-dependent trafficking of the receptors. To test this possibility, we first assessed the level of Tkv receptors at the larval NMJ. Since specific antibodies against Tkv receptors are not available, we expressed an EGFP-tagged Tkv receptor transgene in the motor neurons of σ2-adaptin mutants. Interestingly, we found a significant accumulation of Tkv receptors at the mutant synapses (D42-Gal4, AP2σKG02457/AP2σang7, UAS-tkv-EGFP: 427.5 ± 20.20, p ≤ 0.001) when compared with control (D42-Gal4/UAS-tkv-EGFP: 100 ± 8.49; Fig. 5A–D,I). In order to analyze the subcellular accumulation, we plotted the intensity profiles of Tkv-EGFP and HRP (a presynaptic membrane maker) in single sections of the acquired images. While in control synapses, Tkv localized both at the presynaptic membrane as well as within the bouton; we found a higher intensity peak of Tkv-EGFP at the presynaptic membranes of σ2-adaptin mutants (Fig. 5J–O). Consistent with these observations, we found that knocking down α-adaptin (D42-Gal4>α-adaptin dsRNA; 276.1 ± 14.75, p ≤ 0.001)] or clathrin light chain (Clc; D42-Gal4>Clc dsRNA; 408.61 ± 21.17, p ≤ 0.001) in motor neurons showed significantly increased synaptic Tkv levels (Fig. 5E–I) with intensity profiles similar to that of σ2-adaptin mutants (Fig. 5P–U). In contrast, mutants defective in proteins involved in later stages of endocytosis did not show Tkv accumulation at the presynaptic membrane (Fig. 5I), which is consistent with previous studies (O’Connor-Giles et al., 2008; G Zhao et al., 2015). These data indicate that endocytosis/trafficking of Tkv receptors in σ2-adaptin mutants is severely compromised, leading to their accumulation at the synaptic membranes.
Neuronal reduction of subunits of AP2 complex or clathrin leads to accumulation of synaptic Tkv. A–H, Confocal images of NMJ synapses at muscle 4 NMJ at A2 hemisegment in control, D42-Gal4/UAS-tkv-EGFP (A, B), D42-Gal4, AP2σKG02457/AP2σang7, UAS-tkv-EGFP (C, D), D42-Gal4, tkv-EGFP/α-adaptin dsRNA (E, F), and D42-Gal4, tkv-EGFP/Clc dsRNA (G, H). The neuronal membrane is marked with HRP (magenta), and EGFP fluorescence is shown in grayscale/green. The bouton area is outlined in the gray channel. Scale bar in H represents 5 μm. I, Histogram showing the relative Tkv level normalized to HRP in D42-Gal4/UAS-tkv-EGFP (100 ± 8.489), D42-Gal4, AP2σKG02457/AP2σang7, UAS-tkv-EGFP (427.5 ± 20.2), D42-Gal4, tkv-EGFP/α-adaptin dsRNA (276.1 ± 14.75), D42-Gal4, tkv-EGFP/Clc dsRNA (408.6 ± 21.17), and Synj1/2 (110.1 ± 6.65) synapses. Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. ***p < 0.001; ns, not significant. J–U, A single confocal section of a bouton labeled for Tkv (represented in grayscale) and presynaptic membrane marker HRP (magenta) in D42-Gal4/UAS-tkv-EGFP (J, K) or D42-Gal4, AP2σKG02457/AP2σang7, UAS-tkv-EGFP (M, N), D42-Gal4, tkv-EGFP/α-adaptin dsRNA (P, Q), and D42-Gal4, tkv-EGFP/Clc dsRNA (S, T). Note that the intensity profile plot across bouton (shown in L, O, R, U as a thin line) shows that σ2-adaptin mutant (O), D42-Gal4>α-adaptin dsRNA (R), and D42-Gal4>Clc dsRNA (U) has more Tkv receptors at the membrane compared with control (L). Scale bar in T represents 2.5 μm.
Loss of σ2-adaptin results in decreased levels of recycling endosomal marker Rab11
Receptors are known to be endocytosed, trafficked to early endosomes, and sorted out for recycling back to the membrane or degraded in the lysosomes (Dunn et al., 1989; Gruenberg et al., 1989; Jovic et al., 2010). This process is mediated by the Rab family of small GTPases (Schwartz et al., 2007). Since the ultrastructural analysis of σ2-adaptin mutant synapse showed an accumulation of enlarged synaptic vesicles similar to mutants of the endolysosomal pathway (Shimizu et al., 2003; West et al., 2015; Inoshita et al., 2017), we next asked whether this pathway has any role in the accumulation of Tkv in σ2-adaptin mutants. We found that the synaptic levels of the early endosomal marker, Rab5 (w1118: 100.0 ± 3.0 vs AP2σKG02457/AP2σang7: 97.25 ± 4.81) or the late endosomal marker, Rab7 (w1118: 100.0 ± 5.71 vs AP2σKG02457/AP2σang7: 115.6 ± 8.17) were not altered in σ2-adaptin mutant synapses (Fig. 6A–J). However, we observed reduced synaptic Rab11 levels (w1118: 100.0 ± 5.73 vs AP2σKG02457/AP2σang7: 61.81 ± 7.10, p ≤ 0.001) in σ2-adaptin mutant synapses (Fig. 6K–S). Western blot analysis revealed that total levels of Rab11 protein in σ2-adaptin mutants were not altered compared with w1118 controls (Extended Data Fig. 6-1). Synaptic Rab11 levels were restored to w1118 control levels on neuronal expression of a σ2-adaptin transgene in the σ2-adaptin mutant (D42-Gal4, AP2σKG02457/UAS-AP2σ, AP2σang7:112.9 ± 8.29; Figure 6O,P,S). Taken together, these data indicate that: (1) compromised endocytosis, and possible defective recycling of the endocytosed Tkv receptors result in their accumulation at the synapse, or/and (2) AP2 complex may regulate Tkv sorting possibly through the endosomal pathway.
σ2-adaptin mutant synapses show a reduction in the recycling endosome marker, Rab11. A–D, Confocal images of NMJ synapses at muscle 4 of A2 hemisegment in control (A, B), AP2σKG02457/AP2σang7 (C, D), double immunolabeled with early endosomal marker Rab5 (represented in grayscale/green) and neuronal membrane marker, HRP (magenta). E, Histogram showing the Rab5 level in control (100.0 ± 3.0) and AP2σKG02457/AP2σang7 (97.25 ± 4.81) synapse. Error bar represents SEM; statistical analysis was done using Student’s t test. ns, not significant. F–I, Confocal images of NMJ synapses at muscle 4 of A2 hemisegment in control (F, G), AP2σKG02457/AP2σang7 (H, I), double immunolabeled with late endosomal marker, Rab7 (represented in grayscale/green), and neuronal membrane marker, HRP (magenta). J, Histogram showing the Rab7 level in control (100.0 ± 5.71) and AP2σKG02457/AP2σang7 (115.6 ± 8.17) synapse. Error bar represents SEM; statistical analysis was done using Student’s t test. ns, not significant. K–R, Confocal images of NMJ synapses at muscle 4 of A2 hemisegment in control (K, L), AP2σKG02457/AP2σang7 (M, N), D42-Gal4, AP2σKG02457/UAS-AP2σ, AP2σang7 (O, P), and Rab11ex2/93 Bi (Q, R) double immunolabeled with recycling endosomal marker, Rab11 (represented in grayscale/green), and neuronal membrane marker, HRP (magenta). Scale bar in R represents 3 μm. S, Histogram showing relative Rab11 level normalized to HRP in control (100 ± 5.73), AP2σKG02457/AP2σang7 (61.81 ± 7.11); D42-Gal4, AP2σKG02457/UAS-AP2σ, AP2σang7 (112.9 ± 8.29) and Rab11ex2/93 Bi (14.21 ± 1.57) synapses. Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. ***p < 0.001; ns, not significant. The data supporting that the levels of Rab11 is not altered in σ2-adaptin mutants is provided in Extended Data Figure 6-1.
Extended Data Figure 6-1
σ2 adaptin mutants have normal Rab11 protein levels. A, Western blotting shows the levels of total Rab11 for control, AP2σKG02457/AP2σang7, D42-Gal4, AP2σang7/UAS- AP2σ, AP2σKG02457, and Rab11ex2/93Bi. Note that total Rab11 protein levels are identical across all genotypes except Rab11ex2/93Bi. B, Histogram showing the average levels of Rab11 normalized with internal control tubulin for control (100 ± 4.65), AP2σKG02457/AP2σang7 (102.8 ± 11.07), D42-Gal4, AP2σang7/UAS- AP2σ, AP2σKG02457 (84.45 ± 6.11), and Rab11ex2/93Bi (65.06 ± 7.16). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. *p < 0.05; ns, not significant. Download Figure 6-1, TIF file.
Rab11 mutants phenocopy BMP-signaling and NMJ defects of σ2-adaptin
Rab11 has been shown to regulate BMP signaling at the Drosophila NMJ (Khodosh et al., 2006; Liu et al., 2014). Because σ2-adaptin mutant synapses showed reduced Rab11 levels, we next asked whether Rab11 had a role in regulating Tkv receptor trafficking. We first examined the levels of pMad and Tkv in Rab11 mutants. We found that Rab11 mutants, as well as animals expressing a dominant-negative form of Rab11 in motor neurons, showed accumulation of pMad at the NMJ synapses (w1118: 100 ± 5.87; Rab11ex2/93 Bi: 147.9 ± 8.58, p ≤ 0.01; UAS-YFP-Rab11S25N/+; D42-Gal4/+: 134.2 ± 4.36, p ≤ 0.05; AP2σKG02457/AP2σang7: 218.8 ± 7.5, p ≤ 0.001; Fig. 7A–H,M). We further assessed Tkv receptor levels in these mutant synapses and found that Rab11 mutants also showed increased Tkv levels at the synapse (D42-Gal4, Rab11ex2/tkv-EGFP, Rab1193Bi: 189.5 ± 10.57) compared with control animals (D42-Gal4/tkv-EGFP: 100 ± 8.71). However, the Tkv enrichment pattern was not the same as that in σ2-adaptin mutants (Fig. 7I–L,N). While σ2-adaptin mutants showed Tkv enrichment at the synaptic membranes, Rab11 mutants had punctate Tkv localization within the boutons, but showed no enrichment at the presynaptic membrane (Extended Data Fig. 7-1). This suggests that while Rab11 does not affect the internalization of Tkv, a defective vesicular trafficking pathway in Rab11 mutants probably leads to the enrichment of Tkv receptors in endosome-like compartments.
Rab11 mutants show elevated levels of synaptic pMad and Tkv. A–H, Confocal images of NMJ from muscle 4 at A2 hemisegment in control (A, B), Rab11ex2/93 Bi (C, D), D42-Gal4-driven dominant-negative YFP-Rab11S25N (E, F), and AP2σKG02457/AP2σang7 (G, H) double immunolabeled with pMad (grayscale/green) and a neuronal membrane marker, HRP (magenta) to mark the bouton outline. Scale bar in H represents 5 μm. I–L, Confocal images of NMJ from muscle 4 at A2 hemisegment in D42-Gal4-driven tkv-EGFP (I, J) and D42-Gal4-driven tkv-EGFP, Rab11ex2/Rab1193Bi (K, L) double immunolabeled with Tkv (grayscale/green) and a neuronal membrane marker, HRP (magenta) to mark the bouton outline. Scale bar in L represents 5 μm. M, Histogram showing the levels of pMad normalized with HRP from muscle 4 at A2 hemisegment in control (100 ± 5.87), Rab11ex2/93 Bi (147.9 ± 8.58), D42-driven YFP-Rab11S25N (134.2 ± 4.36), and AP2σKG02457/AP2σang7 (218.8 ± 7.5). The error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001, **p < 0.01, *p < 0.05. N, Histogram showing the relative Tkv level normalized to HRP in D42-Gal4-driven tkv-EGFP (100 ± 8.71), and D42-Gal4, Rab11ex2/tkv-EGFP, Rab1193Bi (189.5 ± 10.57). Error bar represents SEM; statistical analysis was done using Student’s t test. ***p < 0.001. The data supporting that the Tkv receptor is not enriched at the presynaptic membrane in Rab11 mutants is shown in Extended Data Figure 7-1.
Extended Data Figure 7-1
Tkv-EGFP is not localized at the presynaptic membrane in the Rab11 mutants. A–F, A single confocal section of a bouton labelled for Tkv (represented in grayscale) and presynaptic membrane marker HRP (magenta) in D42-Gal4/UAS-tkv-EGFP (A, B) and D42-Gal4/UAS-tkv-EGFP (D, E). The intensity profile across the bouton in D42-Gal4/UAS-tkv-EGFP (F) suggests that Tkv is not enriched at the presynaptic membrane. Download Figure 7-1, TIF file.
Since σ2-adaptin mutants showed reduced Rab11 levels at the NMJ, we next asked whether Rab11 was responsible for the morphologic defects in these mutants. To assess this, we first examined whether reducing Rab11 levels phenocopied σ2-adaptin mutations. Interestingly, we found that Rab11 mutants or neuronal expression of a dominant-negative form of Rab11 (Rab11S25N) indeed phenocopied the NMJ morphologic defects in σ2-adaptin mutants and showed NMJ overgrowth (w1118: 1.56 ± 0.06; Rab11ex2/93 Bi: 2.77 ± 0.11, p ≤ 0.001; UAS-YFP-Rab11S25N/+; D42-Gal4/+: 2.26 ± 0.08, p ≤ 0.001; and AP2σKG02457/AP2σang7: 2.83 ± 0.12, p ≤ 0.001; Fig. 8A–F). Expressing a wild-type or constitutively active form of Rab11, however, did not alter the synaptic morphology (Extended Data Fig. 8-1). We next examined whether neuronal expression of Rab11 could restore the synaptic overgrowth in σ2-adaptin mutants. We found that expressing the wild-type form of Rab11 in σ2-adaptin mutant background (UAS-YFP-Rab11WT/+; D42-Gal4, AP2σang7/AP2σKG02457: 2.83 ± 0.16, ns) did not prevent the synaptic overgrowth (Fig. 8E,F). Moreover, expressing a constitutively active form of Rab11 (Rab11CA; UAS-YFP-Rab11CA/+; D42-Gal4, AP2σang7/AP2σKG02457: 2.5 ± 0.14, ns) failed to prevent the synaptic overgrowth in σ2-adaptin mutants (Extended Data Fig. 8-2). Consistent with this, expression of Rab11WT, Rab11DN, or Rab11CA in σ2-adaptin mutant background did not rescue the functional defects in σ2-adaptin mutant (Extended Data Figs. 8-3, 8-4). Taken together, these results suggest that σ2-adaptin is crucial for the proper localization of Rab11 at the synapses and possibly required for the formation of the Rab11 positive recycling endosomes. We surmise that these recycling endosomes could be essential for targeting and distributing Tkv receptors at the synapses to regulate BMP signaling.
Rab11 mutants phenocopy NMJ structural defects of σ2-adaptin mutation. A–D, Confocal images of NMJ synapses at muscle 6/7 NMJ at A2 hemisegment in control (A), AP2σKG02457/AP2σang7 (B), Rab11ex2/93 Bi (C), and D42-Gal4>YFP-Rab11S25N (D) double immunolabeled with a presynaptic synaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Scale bar in D represents 20 μm. E, Histogram showing average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control (1.56 ± 0.06), Rab11ex2/93 Bi (2.77 ± 0.11), D42-Gal4-driven dominant-negative YFP-Rab11S25N (2.26 ± 0.08), AP2σKG02457/AP2σang7 (2.83 ± 0.12), and UAS-YFP-Rab11WT/+; D42-Gal4, AP2σang7/AP2σKG02457 (2.83 ± 0.16). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001, *p < 0.05; ns, not significant. F, Histogram showing average bouton area from muscle 6/7 NMJ at A2 hemisegment in control (9.97 ± 0.62), Rab11ex2/93 Bi (5.51 ± 0.33), D42-Gal4-driven dominant-negative YFP-Rab11S25N (6.63 ± 0.31), AP2σKG02457/AP2σang7 (3.71 ± 0.2), and UAS-YFP-Rab11WT/+; D42-Gal4, AP2σang7/AP2σKG02457 (5.14 ± 0.17). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ***p < 0.001, *p < 0.05; ns, not significant. Data supporting that expression of wild-type or constitutive active Rab11 does not alter NMJ morphology and does not restore NMJ morphologic defects of σ2-adaptin mutants is shown in Extended Data Figures 8-1 and 8-2, respectively. Data supporting that expression of wild-type or dominant negative Rab11 and constitutive active Rab11 does not restore the functional defects of σ2-adaptin mutants is shown in Extended Data Figures 8-3 and 8-4, respectively.
Extended Data Figure 8-1
Expressing a wild-type or constitutively active form of Rab11 does not alter the synaptic morphology. A–C, Confocal images of NMJ synapses at muscles 6/7 NMJ at A2 hemisegment showing the synaptic growth in control (A), D42-Gal4-driven UAS-Rab11-GFP (B), and D42-Gal4-driven UAS-Rab11Q70L-GFP (C) double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Expressing the wild-type and active form of Rab11 does not show morphological changes at the synapse. Scale bar in C represents 20 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.56 ± 0.06), UAS-Rab11-GFP/+; D42-Gal4/+ (1.56 ± 0.07), and UAS-Rab11Q70L-GFP; D42-Gal4/+ (1.58 ± 0.09). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. Download Figure 8-1, TIF file.
Extended Data Figure 8-2
Constitutively active Rab11 does not restore the NMJ morphological defects of σ2-adaptin mutants. A–D, Confocal images of NMJ synapses at muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in (A) Control animals, (B) D42-Gal4 > Rab11CA, (C) AP2σKG02457/AP2σang7, and (D) D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 double immunolabeled with a presynaptic synaptic vesicle marker, CSP (magenta) and a neuronal membrane marker, HRP (green) to mark the bouton outline. Scale bar in D represents 20 μm. E, Histogram showing average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.05 ± 0.11), D42-Gal4 > Rab11CA (1.4 ± 0.10), AP2σKG02457/AP2σang7 (2.9 ± 0.14), and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 (2.5 ± 0.14). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01; ns, not significant. Download Figure 8-2, TIF file.
Extended Data Figure 8-3
Overexpression of Rab11 does not restore the functional defects of σ2-adaptin. A, Representative traces of mEJP in control, D42 > Rab11DN, D42 > Rab11WT, heteroallelic AP2σKG02457/AP2σang7, D42 > Rab11DN, AP2σKG02457/AP2σang7, and D42 > Rab11WT, AP2σKG02457/AP2σang7 larvae. B, Representative tracesof EJP in control, D42 > Rab11DN, D42 > Rab11WT, heteroallelic AP2σKG02457/AP2σang7, D42 > Rab11DN, AP2σKG02457/AP2σang7, and D42 > Rab11WT, AP2σKG02457/AP2σang7 larvae. C, Quantification of average EJP amplitude in control (44.42 ± 1.93), D42 > Rab11DN (53.89 ± 2.35), D42 > Rab11WT (52.71 ± 2.42), heteroallelic AP2σKG02457/AP2σang7 (43.57 ± 4.52), D42 > Rab11DN, AP2σKG02457/AP2σang7 (38.03 ± 3.60), and D42 > Rab11WT, AP2σKG02457/AP2σang7 (32.92 ± 2.14). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average mEJP amplitude in control (0.56 ± 0.01), D42 > Rab11DN (0.96 ± 0.08), D42 > Rab11WT (0.78 ± 0.05), heteroallelic AP2σKG02457/AP2σang7 (1.19 ± 0.05), D42 > Rab11DN, AP2σKG02457/AP2σang7 (0.13 ± 0.14), and D42 > Rab11WT, AP2σKG02457/AP2σang7 (1.46 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing average mEJP frequency in control (1.38 ± 0.10), D42 > Rab11DN (4.17 ± 0.47), D42 > Rab11WT (3.78 ± 0.31), heteroallelic AP2σKG02457/AP2σang7 (5.01 ± 0.23), D42 > Rab11DN, AP2σKG02457/AP2σang7 (4.33 ± 0.23), and D42 > Rab11WT, AP2σKG02457/AP2σang7 (4.06 ± 0.34). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. F, Quantification of quantal content in control (50.56 ± 5.82), D42 > Rab11DN (53.14 ± 4.91), D42 > Rab11WT (66.11 ± 5.48), heteroallelic AP2σKG02457/AP2σang7 (38.95 ± 3.65), D42 > Rab11DN, AP2σKG02457/AP2σang7 (30.14 ± 3.03), and D42 > Rab11WT, AP2σKG02457/AP2σang7 (25.07 ± 0.93). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. Download Figure 8-3, TIF file.
Extended Data Figure 8-4
Constitutively active Rab11 does not restore the physiological defects of σ2-adaptin mutants. A, Representative traces of mEJP in control, D42-Gal4 > Rab11CA, heteroallelic AP2σKG02457/AP2σang7, and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 larvae. B, Representative traces of EJP in control (w1118), D42-Gal4 > Rab11CA, heteroallelic AP2σKG02457/AP2σang7, and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 larvae. C, Quantification of average mEJP amplitude (mV) in control (0.67 ± 0.04), D42-Gal4 > Rab11CA (1.13 ± 0.17), heteroallelic AP2σKG02457/AP2σang7 (1.05 ± 0.09) and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 (1.30 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average EJP amplitude in control (44.52 ± 1.07), D42-Gal4 > Rab11CA (56.85 ± 4.221), heteroallelic AP2σKG02457/AP2σang7 (35.40 ± 1.925) and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 (41.41 ± 1.340). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing quantal content in the control (63.43 ± 4.81), D42-Gal4 > Rab11CA (26.88 ± 4.42), heteroallelic AP2σKG02457/AP2σang7 (47.08 ± 4.13) and D42-Gal4 > Rab11CA, AP2σang7/AP2σKG02457 (37.38 ± 5.83). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple comparison test. **p < 0.01; ns, not significant. Download Figure 8-4, TIF file.
In conclusion, σ2-adaptin and Rab11 increase Tkv accumulation and BMP signaling to generate similar NMJ phenotypes via independent pathways.
Discussion
Compromised endocytosis not only perturbs synaptic transmission but also has been implicated in deregulating synaptic growth as demonstrated in Endo, Synj, nwk, shi, Clc, brat, and σ2-adaptin mutant NMJs (Rikhy et al., 2002; Verstreken et al., 2002; Verstreken et al., 2003; Choudhury et al., 2016). The underlying molecular mechanism by which these proteins regulate synaptic growth, however, has only been demonstrated for nwk and brat (O’Connor-Giles et al., 2008; Shi et al., 2013). Our previous study on σ2-adaptin mutants showed no change in levels of endocytic proteins like Endo, Synj, and Dyn (Choudhury et al., 2016), prompting us to investigate the role of σ2-adaptin in synaptic growth signaling. Mutations that affect endocytosis, in general, show synaptic overgrowth and increased BMP effector, pMad. The signaling output of growth-regulating pathwaysis often dependent on intracellular traffic that in part is dependent on endocytosis of activated receptors, ultimately impinging on the BMP, JNK, or Wingless pathways (Shi et al., 2013; Deshpande and Rodal, 2016). Here, we show for the first time that σ2-adaptin genetically interacts with BMP signaling pathway at the synapse. Loss of σ2-adaptin leads to accumulation of Tkv receptor at the NMJ. Additionally, we provide evidence that σ2-adaptin regulates the localization of recycling endosomal protein Rab11.
σ2-adaptin genetically interacts with BMP pathway to regulate neuronal BMP signaling
Endosomal trafficking of BMP receptors is a crucial regulatory mechanism that controls synaptic growth (Rodal et al., 2011). Various proteins interact with BMP receptors to facilitate or attenuate the BMP-dependent signaling cascade (McCabe et al., 2004; XW Zhang et al., 2017). Endocytic proteins appear to be fascinating candidates as BMP receptor interactors. Drosophila loss-of-function endocytic mutants correlate with elevated BMP signaling and neuronal overgrowth phenotype (Dickman et al., 2006; Rodal et al., 2011; Deshpande and Rodal, 2016). Consistent with this, we show that increased Tkv levels at the NMJ result in elevated BMP signaling in σ2-adaptin mutants. If the BMP pathway is responsible for the synaptic overgrowth in σ2-adaptin mutants, we reasoned that reducing the levels of BMP signaling components should rescue the NMJ phenotype. In agreement with this, we found that partially reducing BMP receptors Tkv, Wit, and cytosolic co-Smad molecule, Medea, significantly rescues the NMJ defects in σ2-adaptin mutants. Our data reveal that σ2-adaptin genetically interacts with the negative regulator of BMP signaling, the inhibitory Smad, Dad. Transheterozygotes of Dad and σ2-adaptin mutants have increased number of boutons compared with heterozygotes of either mutant alone. Consistent with this inference, neuronal expression of UAS-Dad in σ2-adaptin mutant background significantly reduces the synaptic overgrowth phenotype. However, reducing BMP signaling only partially reduces the bouton size in σ2-adaptin mutants. There could be at least two plausible explanations for the partial rescue of the bouton size: first, removing only one copy of tkv may not be sufficient for rescuing this defect. Since mutating both copies of tkv results in embryonic lethality, we could not test the epistatic interactions by removing both the copies of Tkv in σ2-adaptin mutants. Second, σ2-adaptin may regulate NMJ bouton size through a different signaling pathway, which remains to be elucidated. Overall, our data suggest that σ2-adaptin negatively regulates the BMP growth signaling pathway to attenuate synaptic growth.
σ2-adaptin regulates trafficking of Thickveins at the NMJ
BMP signaling has been extensively studied in the context of neuronal growth in which the activated Tkv is endocytosed and fuse with the early endosomes, where it activates downstream signaling molecules. The signaling is attenuated when these activated receptor-containing vesicles recycle back to the plasma membrane or fuse with lysosomes for degradation (Rodal et al., 2011; Smith et al., 2012). Trafficking of these receptors into and out of such endosomes provides an additional tier for spatial and temporal modulation of signal transduction. The members of the Rab family of small GTPases regulate various stages of endocytosis (Kelly et al., 2012). Our immunocytochemistry data show elevated Tkv receptor levels at the synapses and motor neuron soma (data not shown) of σ2-adaptin mutants. Besides, levels of Rab11 (known for its role in the recycling of Tkv receptor) are reduced by half in σ2-adaptin mutant synapses.
Interestingly, levels of early and late endosomes marked with Rab5 and Rab7, respectively, remain unaffected at the σ2-adaptin mutant synapses. The intensity profile of Tkv and HRP across the bouton shows that σ2-adaptin mutant has a higher intensity of Tkv at the membrane, indicating that a significant proportion of the Tkv receptors are accumulated at the presynaptic membrane. The pattern of Tkv enrichment in σ2-adaptin and Rab11 mutants were distinct. While σ2-adaptin mutant synapses showed Tkv enriched at synaptic membranes, Rab11 mutants had a rather punctate distribution within the bouton. The differential distribution of these proteins could be because of their distinct roles in the neurons. While σ2-adaptin plays a critical role in retrieving the SV-membrane from the presynaptic membrane, Rab11 is a component of the endolysosomal machinery. A clear separation of Tkv signals/pixels distinguishing plasma membrane Tkv from endosomal Tkv is challenging at the NMJ, given the resolution limit of the confocal system. The difference in Tkv distribution in σ2-adaptin and Rab11 mutants also argues for additional pathways, other than Rab11, through which σ2-adaptin may regulate BMP signaling.
Tkv receptors in σ2-adaptin mutants could be accumulated either at the plasma membrane because of inefficient CME or at the early endosomes caused by inefficient recycling. If Tkv were accumulated at the early endosomes, we would expect greater colocalization with Rab5, which is not the case. However, based on reduced Rab11 staining at the σ2-adaptin mutant synapses, we conclude that the portion of the receptors in Rab5 positive early endosomes fail to recycle back to the plasma membrane. This conclusion also fits with previous observations that defective CME results in the accumulation of endosome-like structures in cultured hippocampal neurons (Kononenko et al., 2014) and is substantiated by our electron microscopy data. The link between clathrin-mediated dynamin-dependent endocytosis and BMP signaling is still a contentious topic. A recent study using human umbilical vein endothelial cells (HUVECs) has shown that treating these cells with BMP-9 triggered Caveolin-1 and dynamin-2-mediated endocytosis of its receptor, activin-like kinase 1 (ALK-1). Surprisingly, this ALK-1 endocytosis was not mediated by Clathrin heavy chain (Tao et al., 2020). At the Drosophila NMJ, endocytosis attenuates BMP signaling (O’Connor-Giles et al., 2008), whereas in Drosophila wing discs and intestinal stem cells, endocytosis facilitates the signaling cascade by internalizing Tkv (Gui et al., 2016; Tracy Cai et al., 2019), pointing toward a tissue-specific mechanism. Our results suggest a model where bulk membrane endocytosis is insufficient in removing Tkv from the plasma membrane; besides, the synapses in σ2-adaptin mutants fail to recycle remaining receptors from early endosomes leading to enhanced signaling and NMJ growth defects.
Functional and morphologic aspects of σ2-adaptin-mediated BMP signaling can be discriminated
Morphologic features of synapses often dictate functional outcomes, and physiological analyses of BMP signaling mutants reveal the same. In wit mutants, the size of the NMJ is significantly reduced with concomitant reduced evoked excitatory potentials (Aberle et al., 2002; Marqués et al., 2002). Analyses of tkv and sax, co-Smad Medea, and transcription factor Mad showed that these NMJs have smaller synapses with severe functional deficits (McCabe et al., 2004). Consistent with the role of BMP-signaling in regulating NMJ growth, gbb mutant larvae also exhibit shorter NMJs with severely reduced evoked potentials (McCabe et al., 2003). σ2-adaptin mutant synapses show a modest reduction in evoked potentials, and the protein is dispensable for maintaining basal synaptic transmission (Choudhury et al., 2016). However, a rundown of EJP amplitudes during high-frequency stimulation in synaptic mutants implicated in CME, such as Endo, Synj, and Dap160, show a rapid stimulus-dependent decline in EJP amplitude that recovers following a period of rest after the high-frequency stimulation paradigm (Verstreken et al., 2003; Koh et al., 2004). In our previous study, we reported that σ2-adaptin mutants do not recover from synaptic depression even after the 90s rest after the cessation of high-frequency stimulation (Choudhury et al., 2016). This observation suggested that in addition to its requirement in synaptic membrane retrieval, the σ2-adaptin function is also required during the much slower process of SV trafficking, possibly at one of the rate-limiting steps in SV regeneration. This conclusion is supported by our EM data that shows the accumulation of endosome-like structures more frequently at the mutant synapses when compared with w1118 controls. EJP and high-frequency recordings from σ2-adaptin mutant synapses with one copy of tkv7 did not show any rescue in synaptic function. These data drive the conclusion that partial reduction of BMP pathway components can only rescue morphologic defects in σ2-adaptin mutants but not functional aspects and that morphologic and functional deficits can be discriminated in these mutants. Besides, the partial rescue of bouton size and bouton clustering at the NMJ argues for possible deregulation of multiple signaling pathways in σ2-adaptin mutants that remain to be explored.
Our study uncovers and extends the existing knowledge of synaptic growth signaling and endocytosis. We provide four lines of evidence on the critical role of σ2-adaptin in modulating BMP-dependent synaptic growth signaling at the Drosophila NMJ. First, we show using genetics that the morphologic defects in σ2-adaptin mutant synapses can be partially rescued by introducing a mutant copy of the BMP receptors, tkv, and wit. We also show a direct epistatic interaction between σ2-adaptin and the inhibitory Smad, Dad. Second, using immunohistochemistry, we show that σ2-adaptin mutant synapses accumulate Tkv at the plasma membrane and some of these receptors that are endocytosed and make it to the early endosomes fail to recycle back to the plasma membrane. Third, our electrophysiology data establish that morphologic and functional defects can be discriminated in σ2-adaptin mutants. Finally, our electron micrographs provide evidence for the presence of large endosomes and support our conclusion that σ2-adaptin is critically required at a later step of vesicle regeneration following endocytosis from the plasma membrane.
While this study does not report a direct biochemical or epistatic interaction between σ2-adaptin and Rab11, we observed a significant reduction in Rab11 immunoreactivity in σ2-adaptin mutant synapses that could be restored by neuronal expression of σ2-adaptin transgenes. However, the structural and functional defects of σ2-adaptin mutants could not be restored by neuronal expression of Rab11WT or Rab11CA. This rules out the notion that σ2-adaptin phenotypes result from the observed reduction of Rab11. The Rab11 mutant used in this study is a hypomorph with substantial Rab11 protein being detected by us and others (Khodosh et al., 2006). This precludes further epistatic analyses of this mutant. The fact that Rab11 protein could still be detected in these Rab11 hypomorphic mutants may explain why pMad levels were not as upregulated when compared with σ2-adaptin mutants. Finally, the separation of Tkv immunoreactivity from the synaptic membrane versus that of the endosomal membrane is challenging, given the resolution of the imaging system used here. Our electron micrographs, however, convincingly show an accumulation of endosome-like structures close to the plasma membrane in σ2-adaptin mutant boutons. The endosome-like structures observed in the σ2-adaptin mutants are strikingly similar to structures previously reported for clathrin (Khodosh et al., 2006), AP180 (B Zhang et al., 1998), and Rab11 (Inoshita et al., 2017), among others. Thus, Tkv receptors could likely be enriched in these endosome-like compartments.
We propose a model in which σ2-adaptin/AP2-complex is required for the attenuation of BMP signaling at the Drosophila NMJ. In the absence of AP2, recycling of Tkv is compromised, which results in its enrichment at the presynaptic membrane and/or in the early endosomes leading to elevated BMP signaling and synaptic overgrowth (Fig. 9). This study thus opens new avenues where the role of other CME components and their interaction with various growth signaling pathways can be studied. Since receptor localization and regulation appear to be the central theme in modulating BMP signaling and synapse growth, it will be interesting to perform structure-function analyses of BMP receptors and identify key residues/motifs that interact with AP2 and facilitate its endocytosis. Mutating tyrosine-based signal (YXXϕ) and dileucine-based signal ([DE]XXXL[LI]) motifs in Tkv and Wit could lead to further understanding of these intricate interactions.
Model depicting the role of σ2-adaptin in BMP receptor trafficking at the NMJ. The model depicts a novel function of σ2-adaptin/AP2 in Tkv receptor trafficking at the Drosophila NMJ. Retrograde NMJ growth signaling in Drosophila involves Gbb ligand that is secreted from postsynaptic muscles and binds to BMP receptors on the presynaptic membrane to activate them (Aberle et al., 2002; McCabe et al., 2003). Activated receptors are then internalized through CME and fuse with early endosomes to trigger the downstream signaling cascade (Hartung et al., 2006). From early endosomes, receptors either get sorted to the Rab11-positive recycling endosomes that recycle them back to the presynaptic membrane or are sorted for lysosomal degradation (Liu et al., 2014; Deshpande and Rodal, 2016). Depletion of σ2-adaptin/AP2-complex leads to enrichment of Tkv receptors at the presynaptic membrane and/or in the early endosomes leading to elevated BMP signaling resulting in synaptic overgrowth. It is also likely that reduced synaptic Rab11 levels in σ2-adaptin might perturb Rab11-mediated recycling of Tkv leading to enhanced BMP-signaling.
Acknowledgments
Acknowledgments: We thank Dr. Tsubasa Tanaka for sharing Rab7 and Rab11 antibodies, Dr. Marino Zerial for Rab5 antibodies, and Dr. Peter ten Dijke for pMad antibody. We also thank Dr. Avital Rodal for sharing Rab11 mutants; the Bloomington Drosophila Stock Center (BDSC), Vienna Drosophila RNAi Centre (VDRC), and Drosophila Genomics Resource Centre (DGRC) for fly stocks; Developmental Studies Hybridoma Bank (DSHB), the University of Iowa for monoclonal antibodies; and Dr. Manish Jaiswal for his helpful comments on this manuscript.
Footnotes
The authors declare no competing financial interests.
This work was supported by the Science and Engineering Board (SERB) Project Grant CRG/2021/000599, the Government of India, and by intramural funds from the Indian Institute of Science Education and Research Bhopal (V.K.).
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.