σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila

Abstract

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling.

Keywords: Drosophila; angur; pMAD; physiology; synapse.

Figure 1

angur is an allele of σ₂-adaptin, the smallest subunit of adapter protein 2 (AP2) complex. (A) Deficiency mapping showed the angur locus to be located in the region of 93D₁–93D₄ on the third chromosome of Drosophila. The three deficiency lines that uncovered the angur locus—Df(3R)BSC677, Df(3R)ED10838, and Df(3R) ED10845—are labeled. Solid bars mark the deleted regions in these deficiency lines. The 93D₁–93D₄ region of the chromosome contains at least 10 identified genes. Different colored arrows represent the relative positions and orientations of the genes in the 93D₁–93D₄ region. While angur⁷ and angur² were obtained from mobilization of EP0877, angur⁵ was obtained from mobilization of KG06118b. The P-element in angur alleles was located in the 5′ end of the hsrω gene but did not affect expression of the hsrω gene. (B) Genomic organization of the σ₂-adaptin locus showing exons (represented by solid boxes) and introns (represented by thin lines). The transcription start site is represented by an arrow, and the untranslated regions are shown by black solid boxes. The insertion site of P-element KG02457 lies in the third exon of the gene. (C) Semiquantitative (top panel) and quantitative RT-PCR (bottom panel) depicting transcript levels of σ₂-adaptin in controls, angur⁷/AP2σ^KG02457, angur⁷/angur², angur⁷/angur⁵, actin5C-Gal4-driven σ₂-adaptin RNAi, and rescued heteroallelic mutant (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457), respectively. σ₂-adaptin transcript level is dramatically reduced in the heteroallelic mutants or actin5C-Gal4-driven σ₂-adaptin RNAi. rp49 transcript level was used as an internal concentration control for messenger RNA (mRNA). The error bars in the bottom panel represent SEM. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. (D) Rescue of AP2σ^KG02457 mutants and heteroallelic combinations in homozygous or transheterozygous combinations using ubiquitous actin5C-Gal4 or the neuronal elav^C155-Gal4 drivers.

Figure 2

angur mutant eye clones show severe defects in rhabdomere development. (A–C) Brightfield images of (A) control (FRT82B, angur⁷), (B) mutant angur⁷ eye clone (EGUF/+; FRT82B, angur⁷/FRT82B, GMR-hid), and (C) transgene-rescued animal (EGUF/+; FRT82B, angur⁷, UAS-AP2σ/FRT82B, GMR-hid). (D–F) SEMs of (D) control (FRT82B, angur⁷), (E) mutant angur⁷ eye clone (EGUF/+; FRT82B, angur⁷/FRT82B, GMR-hid), and (F) transgene-rescued animal (EGUF/+; FRT82B, angur⁷, UAS-AP2σ/FRT82B, GMR-hid). (G–I) TEMs of (G) control (FRT82B, angur⁷), (H) mutant angur⁷ eye clone (EGUF/+; FRT82B, angur⁷/FRT82B, GMR-hid), and (I) transgene-rescued animal (EGUF/+; FRT82B, angur⁷, UAS-AP2σ/FRT82B, GMR-hid). Scale bar, 2 μm. (J) ERG traces of control animals (FRT82B, angur⁷) shown in blue, mutant angur⁷ eye clone animals (EGUF/+; FRT82B, angur⁷/FRT82B, GMR-hid) shown in red, and transgene-rescued animal (EGUF/+; FRT82B, angur⁷, UAS-AP2σ/FRT82B, GMR-hid) shown in green. (K) Histogram showing quantification of receptor potential amplitudes of the preceding genotypes. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. Note that compared to the mutant eye clones, the rescued animals showed mild but significant rescue of receptor potential depolarization.

Figure 3

Mutation in σ₂-adaptin causes aberrant neuromuscular junction formation. (A–D) Confocal image of NMJ synapses at muscle 4 of (A) control animals, (B and C) heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457 and angur⁷/angur⁵), and (D) transgene-rescued animal (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) double immunolabeled with a presynaptic marker CSP (green) and neuronal membrane marker HRP (red) to reveal the bouton outline. Compared to the control NMJ, the heteroallelic combination of σ₂-adaptin shows severely altered NMJ morphology. Scale bar, 15 μm. (E–J) Confocal images of NMJ synapses at muscle 4 co-labeled with CSP (green) and HRP (red) of (E) control and (F–J) neuronally expressing RNAi against AP2 complex subunits or PI4KIIIα: (F) elav^C155/+; α-adaptin RNAi/+, (G) elav^C155/+; β₂-adaptin RNAi/UAS-Dicer, (H) elav^C155/+; μ₂-adaptin RNAi/UAS-Dicer, (I) elav^C155/+; σ₂-adaptin RNAi/UAS-Dicer, and (J) elav^C155/+; PIK4IIIαRNAi/+. Note that neuronal knockdown of any of the subunits of the AP2 complex or PIK4IIIα phenocopies σ₂-adaptin mutations. Scale bar, 15 μm. (K) Histogram showing average number of boutons at muscles 6/7 of the A2 hemisegment in control animals (98.2 ± 4.8), heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457: 185.4 ± 6; angur⁷/angur²: 233.4 ± 7; angur⁷/angur⁵: 274 ± 8.5) and the rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 89.9 ± 4.0). The numbers in columns represent the number of 6/7 NMJs of the A2 segment used for bouton quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (L) Histogram showing average bouton area in control animals (8.7 ± 0.5 μm²), heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457: 2.3 ± 0.13 μm²; angur⁷/angur²: 3.5 ± 0.12 μm²; angur⁷/angur⁵: 2.5 ± 0.1 μm²) and rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 6.4 ± 0.4 μm²). The numbers in columns represent the number of boutons used for quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (M) Histogram showing quantification of average bouton numbers at muscles 6/7 of the A2 hemisegments of third instar larvae in control animals and neuronally expressing RNAi against AP2 subunits (control at 18°, 85 ± 3; control at 28°, 98 ± 2; α-adaptin, 167 ± 13; β₂-adaptin, 145 ± 7.0; μ₂-adaptin, 137 ± 5; σ₂-adaptin, 118 ± 4) or PI4KIIIα (144 ± 7). At least 16 NMJ synapses of muscles 6/7 of the A2 hemisegment of each genotype were used for counting the number of boutons. ***P < 0.0001. The numbers in the columns represent the number of animals used for quantification. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test.

Figure 4

σ₂-adaptin facilitates basal synaptic transmission as well as synaptic vesicle endocytosis. (A) Representative images of FM1-43 dye uptake by control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457), and transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) synapses. (B) Relative synaptic FM1-43 fluorescence uptake in control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457: 72.1 ± 2.3% and angur⁷/angur⁵: 49.73 ± 1.7%) and transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 82.1 ± 2.6%) synapses. ***P < 0.0001; **P < 0.001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (C) Representative traces of mEJP in control animals, heteroallelic σ₂-adaptin mutant combinations (angur⁷/AP2σ^KG02457 and angur⁷/angur⁵), transgenic-rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457), and elav^C155/+; α-adaptin RNAi/+ animals. (D) Representative traces of EJPs in control animals, σ₂-adaptin heteroallelic mutant combinations (angur⁷/AP2σ^KG02457 and angur⁷/angur⁵), transgene-rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457), and elav^C155/+; α-adaptin RNAi/+ animals. (E) Histogram showing average mEJP amplitude in control animals (0.65 ± 0.03 mV), heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457: 0.91 ± 0.08 mV; angur⁷/angur⁵: 0.85 ± 0.04 mV), transgene-rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 0.69 ± 0.04 mV), and elav^C155/+; α-adaptin RNAi/+ (0.94 ± 0.06 mV) animals. At least 8 NMJ recordings of each genotype were used for quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (F) Histogram showing average EJP amplitude in control animals (43.25 ± 1.67 mV), heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457: 31.60 ± 1.9 mV; angur⁷/angur⁵: 28.34 ± 1.05 mV), transgene-rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 42.06 ± 1.14 mV), and elav^C155/+; α-adaptin RNAi/+ (25.17 ± 2.24 mV) animals. At least 8 NMJ recordings of each genotype were used for quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (G) Quantification of quantal content in control animals (67.63 ± 3.37), heteroallelic σ₂-adaptin mutants (angur⁷/AP2σ^KG02457: 35.48 ± 2.45; angur⁷/angur⁵: 34.54 ± 2.25), transgene-rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 64.54 ± 5.18), and elav^C155/+; α-adaptin RNAi/+ (27.26 ± 2.53) animals. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (H) Graph showing the SpH fluorescence response to a 50-Hz stimulus train for 10 sec in 2.0 mM Ca²⁺ containing HL3 of control (blue, n = 37 boutons, 5 animals; elav^3E1, UAS-SpH/+), mutant (orange, n = 33 boutons, 5 animals; elav^3E1, UAS-SpH, AP2σ^KG02457/ang⁷), and rescued (green, n = 29 boutons, 5 animals; elav^3E1, UAS-SpH, AP2σ^KG02457/ang⁷, UAS-AP2σ) synapses. The pink horizontal bar represents the stimulation time. Tau values were calculated after 10 sec of stimulation by fitting the fluorescence decay curve to one-phase exponential. σ₂-adaptin mutant (7.60 ± 0.17) shows a mild but significant difference in SpH kinetics compared to control (6.91 ± 0.17) and rescue (6.60 ± 0.23).

Figure 5

Reducing σ₂-adaptin levels in neurons causes temperature-sensitive paralysis at elevated temperature. (A and B) Control (elav^C155/+) 2- to 3-day-old adult flies do not suffer paralysis in 3 min at 38^ο, whereas similarly aged adult flies expressing σ₂-adaptin RNAi (elav^C155/+; σ₂-adaptin RNAi/+) in neurons showed complete paralysis in 3 min at 38^ο. These animals recovered within 2 min when shifted to 25^ο (not shown). (C) Paralysis profile of control animals and animals expressing RNAi against σ₂-adaptin in neurons (elav^C155/+; σ₂-adaptin RNAi/+) and shi^ts2/+. While the control animals do not suffer paralysis in 3 min, the RNAi-expressing animals are paralyzed above 35^ο. Five trials were performed for each genotype at each temperature in a sushi cooker (Ramaswami et al. 1993). Each trial contained 10–15 animals. Error bars represent standard error of the mean. (D–I) Representative traces of EJPs under high-frequency stimulation of indicated genotypes stimulated at 10 Hz for 5 min at 34° in 1.5 mM Ca²⁺ containing HL3. Unlike shibre^ts1 (G), mutant (E) and neuronally reduced α- or σ₂- adaptin (H and I) animals do not show complete reduction in EJP amplitude at the end of 5 min of stimulation.

Figure 6

σ₂-adaptin function is essential under high-frequency stimulation to regenerate synaptic vesicles. (A) Normalized EJP amplitudes in control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457and angur⁷/angur⁵), rescued larvae (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457), and neuronally knocked down α-adaptin (elav^C155/+; α-adaptin RNAi/+) animals stimulated at 10 Hz for 5 min in 1.5 mM Ca²⁺. The mutant larvae showed stimulus-dependent decline in the EJP amplitudes over time. (B) Representative traces of indicated genotypes under high-frequency stimulation at 10 Hz for 5 min in 1.5 mM Ca²⁺ containing HL3. After 1.5 min of rest, test stimuli were given every 30 sec to assess the recovery of EJP from synaptic depression. Note that the mutant synapses do not recover to the first stimulus levels even after 4 min of rest from high-frequency stimulation. (C) Estimation of endo-exo cycling pool at NMJ synapses of indicated genotypes before high-frequency stimulation and after 4 min of rest from high-frequency stimulation. While the controls and rescue synapses maintained the ECP pool to its initial value after 4 min of rest, the σ₂-adaptin mutant synapses and the elav^C155/+; α-adaptin RNAi/+ synapses show significant decline in the endo-exo cycling pool and do not recover to the initial value after 4 min of rest. n = 50 boutons from eight different NMJs of muscles 6/7 of the A2 hemisegment were counted. (D) Synaptic depression curve showing the depletion in quantal content over stimulus number in synapses stimulated at 3 Hz in the presence of 1 μM Bafilomycin A1 to deplete ECP. (E) Histogram showing ECP estimates of control (25,050 ± 1511), heteroallelic mutants (angur⁷/AP2σ^KG02457: 16,590 ± 974; angur⁷/angur⁵: 15,040 ± 870), elav^C155/+; α-adaptin RNAi/+ (15,730 ± 1031), and rescued animals (actin5C-GAL4/+; angur⁷/UAS-AP2σ, AP2σ^KG02457: 23,635 ± 1358) obtained from Y-intercept of regression, calculated by back-extrapolating from stimulus numbers 3800–6000 on a cumulative plot of Figure 6D. **P ≤ 0.0006; *P ≤ 0.0007. Numbers in the columns represent the number of animals used for experiments and quantification. The error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (F) Synaptic depression curve showing the depletion of the total vesicle pool over stimulus number estimated by stimulating the synapses at 10 Hz in presence of 1 μM Bafilomycin A1. (G) Histogram showing the estimation of total vesicle pool of control animals (94,610 ± 5929), heteroallelic mutants (angur⁷/AP2σ^KG02457: 53,220 ± 3156; angur⁷/angur⁵: 50,930 ± 2877), elav^C155/+; α-adaptin RNAi/+ (63,460 ± 3650), and the rescued animals (actin5C-GAL4/+;angur⁷/UAS-AP2σ, AP2σ^KG02457: 102,200 ± 6315) calculated by integrating quantal content over stimulus number in synaptic depression plot of Figure 6F until depletion was achieved. ***P < 0.0001. Numbers in the columns represent the number of animals used for experiments and quantification. The error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test.

Figure 7

σ₂-adaptin is required specifically for AP2 complex and clathrin stability. (A–E) Representative images of third instar larval boutons from control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457), and transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) NMJ synapses co-labeled with HRP (red) and other synaptic proteins (green): (A) α-adaptin; (B) β₂-adaptin; (C); EYFP-Clc (D) Endophilin 1, and (E) Dynamin 1. The histograms on the right show quantification of these synaptic proteins in the boutons, expressed as percentage of control levels. The fluorescence intensity of at least 50 individual boutons was measured and the background subtracted for the quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (F) Western blot analysis of various synaptic proteins in larval brain of control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457 and angur⁷/angur⁵), and rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457). Level of α-actin was used as loading control. The level of clathrin was assessed by probing the blots with anti-GFP antibody. For the EYFP-Clc experiment, the control genotype was elav^C155/+; EYFP-Clc/+; the heteroallelic mutant genotype was elav^C155/+; EYFP-Clc/+; angur⁷/AP2σ^KG02457; and the rescue genotype was elav^C155/+; EYFP-Clc/+; angur⁷/UAS-AP2σ, AP2σ^KG02457.

Figure 8

Mutations in σ₂-adaptin cause disruption of the presynaptic cytoskeleton and upregulated BMP signaling. (A–F) Representative images of third instar larval NMJs from (A and B) control animals, heteroallelic σ₂-adaptin mutants (C and D) (angur⁷/AP2σ^KG02457), and transgene-rescued (E and F) (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) animals co-labeled with HRP (red) and 22C10 (green). (G) Histogram showing quantification of the percentage of boutons containing Futsch loops of the indicated genotypes. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. n = 6 NMJ synapses of muscles 6/7 of the A2 hemisegment. (H) Representative images of third instar larval boutons from (a) control, (b) heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457), and (c) transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) NMJ synapses co-labeled with HRP (red) and pMAD (green). (I) Histogram showing quantification of synaptic pMAD fluorescence of the indicated genotypes. The fluorescence intensity of at least 50 individual boutons was measured and the background subtracted for the quantification.***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (J) Representative images of motor nuclei of third instar larval VNC from (a) control, (b) heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457), and (c) transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) animals co-labeled with Elav (red) and pMAD (green). (K) Histogram showing quantification of the nuclear pMAD fluorescence of the indicated genotypes. The fluorescence intensities of at least 50 motor nuclei were measured both for Elav and for pMAD, and the ratio of pMAD/elav represented in the quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test.