Deployment of endocytic machinery to periactive zones of nerve terminals is independent of active zone assembly and evoked release
- Department of Neurobiology, Harvard Medical School, United States
- Department of Neuroscience and Institute for Translational Neuroscience, New York University Grossman School of Medicine, United States
- Department of Biology, Brandeis University, United States
Figures
Figure 1 with 4 supplements
Deployment of endocytic proteins after chronic silencing or acute depolarization of mouse hippocampal neurons.
(A) Schematic of the experiment in mouse hippocampal neurons. For chronic silencing, a cocktail (‘blockers’) with tetrodotoxin (TTX, 1 μM final concentration), ω-agatoxin IVA (ω-Aga, 250 nM), and ω-conotoxin GVIA (ω-Cono, 200 nM) was added every 3 days starting DIV3. For acute depolarization (‘KCl’), neurons were stimulated with 50 mM KCl for 30 s before fixation. (B–I) Example side-view synapses and average line profiles for neurons stained for Bassoon, Synaptophysin, and Amphiphysin (B, C), PIPK1γ (D, E), or AP-180 (F, G), or Synaptophysin, Munc13-1, and Dynamin-1 (H, I). Neurons were stained for a protein of interest (Amphiphysin, PIPK1γ, AP-180, or Dynamin-1; imaged in STED), an active zone marker (Bassoon or Munc13-1; imaged in STED), and Synaptophysin (imaged in confocal). An area of interest was positioned perpendicular to the center of the active zone marker, and synapses were aligned via the peak fluorescence of the active zone marker in the average profiles (C, E, G, I). Line profile plots were normalized to the average signal in the untreated condition. Dashed lines in C, E, G, and I mark average levels in the untreated condition, and gray shaded areas represent the active zone and periactive zone area; n in B, C (synapses/cultures): untreated 38/3, blockers 40/3, KCl 45/3; D, E: untreated 51/3, blockers 49/3, KCl 42/3; F, G: untreated 58/3, blockers 61/3, KCl 50/3; H, I: untreated 45/3, blockers 43/3, KCl 42/3. (J, K) Quantification and statistical analyses of the experiment shown in B–I, including peak-to-peak distance of the active zone marker and the protein of interest (J), and of the peak levels in the periactive zone area (K). The periactive zone area is defined as an area within 68 nm on each side of the peak of the active zone marker (gray shaded areas in C, E, G, I); n as in B–I. (L–Q) Example en-face synapses (L–O) and quantification of the number of objects (P) and distance of these objects to the center of the active zone marker (Q) of the experiment shown in A–K; n in P, Q (synapses/cultures): Amphiphysin, untreated 20/3, blockers 21/3, KCl 21/3; PIPK1γ, untreated 23/3, blockers 21/3, KCl 20/3; AP-180, untreated 21/3, blockers 24/3, KCl 18/3; Dynamin-1, untreated 22/3, blockers 20/3, KCl 22/3. Data are shown as mean ± SEM; *p < 0.05, **p < 0.05, ***p < 0.001 shown compared to the untreated condition determined by Kruskal–Wallis followed by Holm post hoc tests (J for Amphiphysin, PIPK1γ, and AP-180; K, P, Q), or by one-way ANOVA followed by a Tukey–Kramer post hoc test (J for Dynamin-1). For quantification of confocal images, see Figure 1—figure supplement 1; for a workflow of STED analyses, see Figure 1—figure supplement 2; for assessment of AP-180 using an independent antibody, see Figure 1—figure supplement 3, for additional analyses of en-face synapses, see Figure 1—figure supplement 4.
Figure 1—figure supplement 1
Confocal microscopic analyses of synapses after chronic silencing or acute depolarization of mouse hippocampal neurons.
(A, B) Example confocal images (A) and quantification of the average intensities (B) of Amphiphysin, PIPK1γ, AP-180, Dynamin-1, Bassoon, and Munc13-1 at synapses identified as Synaptophysin puncta. Intensities are normalized to the average signals in the untreated conditions per culture; n in B (images/cultures): Amphiphysin, 14/3; PIPK1γ, untreated 14/3, blockers 14/3, KCl 13/3; AP-180, 15/3; Dynamin-1, 14/3; Synaptophysin, untreated 57/3, blockers 57/3, KCl 56/3; Bassoon, untreated 43/3, blockers 43/3, KCl 42/3; Munc13-1, 14/3. The increase in Munc13-1 upon chronic silencing, which we previously reported (Held et al., 2020), and of Synaptophysin, may reflect a homeostatic adaptation. The decrease in Amphiphysin upon KCl stimulation may reflect a redistribution of this protein during prolonged stimulation. Data are mean ± SEM; *p < 0.05, **p < 0.05, ***p < 0.001 compared to the untreated condition determined by one-way ANOVA followed by a Tukey–Kramer post hoc tests for Bassoon or Kruskal–Wallis followed by Holm post hoc tests for Amphiphysin, PIPK1γ, AP-180, Dynamin-1, Synaptophysin, and Munc13-1.
Figure 1—figure supplement 2
Workflows for STED analyses in mouse hippocampal neurons and for confocal analyses at Drosophila neuromuscular junctions.
(A) Workflow for the analyses of side-view synapses of mouse hippocampal neurons, showing an example synapse immunostained for the active zone marker Bassoon (imaged in STED), PIPK1γ (imaged in STED), and Synaptophysin (imaged in confocal). Synapse selection and placement of an area of interest (white rectangle with line profile direction indicated by arrow) perpendicular to the marker (Bassoon, in this example) is done by an experimenter blind for the protein of interest (PIPK1γ, in this example). Next, the protein of interest channel is activated, and the profile is generated. Finally, the average line profiles, peak intensities, and distance of proteins of interest to the marker are plotted. (B) Workflow for the analyses of en-face synapses of mouse hippocampal neurons, showing an example synapse immunostained for Bassoon (imaged in STED), PIPK1γ (imaged in STED), and Synaptophysin (imaged in confocal). First, synapse selection is done by an experimenter blind for the protein of interest. Next, the channel of the protein of interest is activated, and objects containing endocytic proteins and the marker are identified in the respective channels using an algorithm. Finally, the number of objects per synapse, their lateral distance to the active zone, and their integrated intensity are plotted. (C) Workflow for analyses of Drosophila neuromuscular junctions. Terminals are analyzed both in 3D and in 2D half-maximum intensity projections. First, the average intensities of the active zone marker (Brp in this example) and the endocytic protein (Nervous Wreck in this example) are quantified in the full 3D volume of the terminal. Next, the periactive zone levels and degree of polarization are analyzed in 2D half-maximum intensity projections. The polarization of each protein is quantified as the ratio between its average intensity at the mesh over its average intensity in the core. To conduct this analysis, segmentation into mesh and core is performed based on the difference in signal between proteins enriched in the periactive zone mesh (e.g. Nwk and Dynamin) vs. proteins enriched in the core region (e.g. Brp and Pak) as described in the methods. White lines delineate the center of the mesh regions. The mesh is ~200 nm wide, and the core is the remaining enclosed region within the innermost bounds. The resulting regions of interest (ROIs) are used to measure average intensities within the mesh and the core and its ratio.
Figure 1—figure supplement 3
Assessment of AP-180 with alternate antibody after chronic silencing or acute depolarization of mouse hippocampal neurons.
(A, B) Example side-view synapses (A) and average line profiles of AP-180 (antibody A246) and Munc13-1 (B). Neurons were stained for AP-180 (imaged in STED), Munc13-1 (imaged in STED), and the synaptic vesicle marker Synaptophysin (imaged in confocal). An area of interest was positioned perpendicular to the center of the Munc13-1 object, and synapses were aligned via the peak fluorescence of Munc13-1 in the average profiles. Line profiles were normalized to the average signal in the untreated condition. Dashed lines mark average levels in the untreated condition, and gray shaded areas represent the active zone area; n in B (synapses/cultures): untreated, 58/3; blockers, 61/3; KCl, 50/3. (C, D) Quantification of the peak-to-peak distance of the active zone marker and the protein of interest (C), and of the peak levels in the periactive zone area (D). The periactive zone area is defined as an area within 68 nm on each side of the peak of the active zone marker (gray shaded areas in B); n as in B. Data are mean ± SEM; *p < 0.05, ***p < 0.001 compared to the untreated condition determined by Kruskal–Wallis followed by Holm post hoc tests.
Figure 1—figure supplement 4
Additional analyses of en-face synapses after chronic silencing or acute depolarization of mouse hippocampal neurons.
Quantification of the average integrated intensities (calculated as the object area multiplied by its average fluorescence intensity) of the Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 objects detected in en-face synapses from Figure 1L–Q. Intensities are normalized to the average signals in the untreated conditions per culture; n as in Figure 1P. Data are mean ± SEM; **p < 0.01, ***p < 0.001 compared to the untreated condition determined by one-way ANOVA followed by a Tukey–Kramer post hoc test for AP-180 or Kruskal–Wallis followed by Holm post hoc tests for Amphiphysin, PIPK1γ, and Dynamin-1.
Figure 2 with 1 supplement
Localization of endocytic proteins at Drosophila NMJs relative to Synapsin or Nervous Wreck.
(A, B) Example boutons of Drosophila NMJs stained for Nervous Wreck and either EndoA, Dap160, Dynamin, or Synapsin, and quantification of the co-localization between Nervous Wreck and these proteins measured as the Pearson’s coefficient; n (NMJ/animals): EndoA 11/3, Dap160 10/3, Dynamin 11/3, Synapsin 11/3. (C, D) Same as A, B but relative to Synapsin instead of Nervous Wreck; n (NMJ/animals): EndoA 11/3, Dap160 11/3, Dynamin 10/3. Data are shown as mean ± SEM. Images acquired by STED microscopy. For antibody validation, see Figure 2—figure supplement 1.
Figure 2—figure supplement 1
Validation of EndoA, Dap160, and Dynamin antibodies in Drosophila NMJs.
Example confocal images and quantification of the signal of EndoA (A, B), Dap160 (C, D), or Dynamin (E, F) in NMJs expressing either driver alone (control) or an RNAi against the indicated gene. Data are expressed as the percentage of the control; n in B (NMJs/animal): control 15/3, EndoA-RNAi 14/3; (D) control 3/2, Dap160-RNAi 3/2; (F) control 19/6, Dyn-RNAi 38/6. Data are mean ± SEM.
Figure 3 with 1 supplement
Deployment of endocytic proteins after chronic silencing or acute stimulation of Drosophila NMJs.
(A) Schematic of the experiment at the Drosophila neuromuscular junction (NMJ). To silence neurons chronically, tetanus neurotoxin light chain (‘TeNT’) was expressed in Type 1b motor neurons on muscle 1 using the GAL4 UAS system. These neurons were compared to those from larvae expressing the M11b-GAL4 driver alone (‘control’). To activate neurons, electrical stimulation was applied at 40 Hz for 3 min (‘40 Hz’) to Type 1b motor neurons on muscles 4 and 6/7, and comparisons were made to the contralateral, unstimulated side (‘no stim’). (B–G) Example maximum intensity projections of ventral half of individual boutons with or without TeNT-expression stained for Brp, Dynamin and Nervous Wreck or Brp, EndoA, and Dap160 (B), and quantification of the number of Brp objects per µm2 of NMJ (C) and their integrated intensity (D). For Brp, Dynamin, Nervous Wreck, EndoA, and Dap160, the average fluorescence intensity per bouton (E), the average intensity at the periactive zone mesh (F) and the polarization within periactive zone units (G) were quantified; n in C,D (NMJs/larvae): control 28/5, TeNT 27/6; E for Nervous Wreck, Dynamin, and Brp: control 28/5, TeNT 28/6; E for Dap160 and EndoA: control 19/6, TeNT 18/6; F,G for Nervous Wreck, Dynamin, and Brp: control 28/5, TeNT 22/6; F, G for Dap160 and EndoA: control 19/6, TeNT 17/6. (H–M) Same as B–G but comparing acutely stimulated (‘40 Hz’) and unstimulated terminals (‘no stim’) boutons; n in I, J: no stim 26/14, 40 Hz 24/14; K for Nervous Wreck, Dynamin, and Brp: no stim 26/14, 40 Hz 25/14; K for Dap160 and EndoA: no stim 13/7, 40 Hz 13/7; L, M for Nervous Wreck, Dynamin and Brp: no stim 26/14, 40 Hz 23/14; L, M for Dap160 and EndoA: no stim 13/7, 40 Hz 13/7. Data in D–F and J–L are normalized to the average of the control condition. Data are mean ± SEM; *p < 0.05 determined by two-sided Student’s t-tests (E for Nervous Wreck, EndoA, and Dap160; F for EndoA and Dap160; G for Brp, Nervous Wreck, EndoA, and Dap160; I–L for EndoA and Dap160; M for Brp, Dynamin, and Nervous Wreck) or two-sided Mann–Whitney U tests (C–E for Brp and Dynamin; F, G for Dynamin; L, M for EndoA and Dap160). Images acquired by Airyscan microscopy, for quantification of EndoA and Dap160 using STED microscopy, see Figure 3—figure supplement 1.
Figure 3—figure supplement 1
Assessment of EndoA and Dap160 after chronic silencing of Drosophila NMJs using STED microscopy.
(A-D) Example boutons with or without TeNT-expression (A), and quantification of the average fluorescence intensity of EndoA and Dap160 per bouton (B), average intensity at the periactive zone mesh (C) and the polarization within periactive zone units (D). Data in B and C are normalized to the average of the control condition; n in B (NMJ/animal): control 20/6, TeNT 16/6; (C, D) control 20/6, TeNT 15/6. Data are mean ± SEM; *p < 0.05 determined by two-sided Student’s t-tests. Images acquired by STED microscopy.
Figure 4 with 3 supplements
Deployment of endocytic proteins after CaV2 ablation in mouse hippocampal neurons.
(A) Schematics of the Cacna1a, Cacna1b, and Cacna1e mutant alleles that constitute the conditional CaV2 triple knockout mouse line as described in Held et al., 2020. (B–I) Example side-view synapses and average line profiles of Amphiphysin and Bassoon (B, C), PIPK1γ (D, E), AP-180 (F, G), and Dynamin-1 and Munc13-1 (H, I). Neurons were stained for a protein of interest (Amphiphysin, PIPK1γ, AP-180, or Dynamin-1; imaged in STED), an active zone marker (Bassoon or Munc13-1; imaged in STED), and Synaptophysin (imaged in confocal). An area of interest was positioned perpendicular to the center of the active zone marker, and synapses were aligned via the peak fluorescence of the active zone marker in the average profiles (C, E, G, I). Line profile plots were normalized to the average signal in the controlCav2 condition. Dashed lines in C, E, G, and I mark average levels in the controlCav2 condition, and gray shaded areas represent the active zone and periactive zone area; n in B, C (synapses/cultures): controlCav2 58/3, cTKOCav2 50/3; D, E: controlCav2 50/3, cTKOCav2 49/3; F, G: controlCav2 50/3, cTKOCav2 47/3; H, I: controlCav2 48/3, cTKOCav2 50/3. (J, K) Quantification and statistical analyses of the experiment shown in B–I, including peak-to-peak distance of the active zone marker and the protein of interest (J), and of the peak intensity in the periactive zone area (K). The periactive zone area is defined as an area within 68 nm on each side of the peak of the active zone marker (gray shaded areas in C, E, G, I); n as in B–I. (L–Q) Example en-face synapses (L–O) and quantification of the number of objects (P) and distance of these objects to the center of the active zone marker (Q) of the experiment shown in B–L; n in P, Q: Amphiphysin, controlCav2 23/3, cTKOCav2 24/3; PIPK1γ, controlCav2 22/3, cTKOCav2 25/3; AP-180, controlCav2 19/3, cTKOCav2 20/3; Dynamin-1, controlCav2 23/3, cTKOCav2 20/3. Data are mean ± SEM; *p < 0.5 determined by two-sided Student’s t-tests (J for Dynamin-1; K for Dynamin-1; P for PIPK1γ and Dynamin; Q for Amphiphysin, AP-180, and Dynamin-1) or two-sided Mann–Whitney U tests (J for Amphiphysin, PIPK1γ, and AP-180; K for Amphiphysin, Bassoon, PIPK1γ, AP-180, and Munc13-1; P for Amphiphysin and AP-180; Q for PIPK1γ). For quantification of confocal signals, see Figure 4—figure supplement 1; for assessment of AP-180 using an independent antibody, see Figure 4—figure supplement 2; for additional assessment of en-face synapses, see Figure 4—figure supplement 3.
Figure 4—figure supplement 1
Confocal microscopic analyses of synapses after CaV2 ablation in mouse hippocampal neurons.
(A, B) Example confocal images (A) and quantification of the average intensities (B) of Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 at synapses identified as Synaptophysin puncta. Intensities are normalized to the average signals in the controlCav2 condition per culture; n in B (images/cultures): Amphiphysin 20/3, PIPK1γ 20/3, AP-180 17/3, Dynamin-1 18/3, Bassoon 57/3, Munc13-1 18/3, Synaptophysin 75/3. Data are mean ± SEM; *p < 0.05 determined by two-sided Student’s t-tests for Amphiphysin, PIPK1γ, Bassoon, and Munc13-1, or two-sided Mann–Whitney U tests for AP-180, Dynamin-1, and Synaptophysin.
Figure 4—figure supplement 2
Assessment of AP-180 with an alternate antibody after CaV2 ablation in mouse hippocampal neurons.
(A, B) Example side-view synapses (A) and average line profiles (B) of AP-180 (antibody A246) and Munc13-1. Neurons were stained for AP-180 (imaged in STED), Munc13-1 (imaged in STED), and the synaptic vesicle marker Synaptophysin (imaged in confocal). An area of interest was positioned perpendicular to the center of the Munc13-1 object, and synapses were aligned via the peak fluorescence of Munc13-1 in the average profiles. Line profiles were normalized to the average signal in the controlCav2 condition. Dashed lines mark average levels in the controlCav2, and gray shaded areas represent the active zone area; n in B (synapses/cultures): controlCav2 50/3, cTKOCav2 48/3. (C, D) Quantification of the peak-to-peak distance of Munc13-1 and AP-180 (C) and of their peak levels in the periactive zone area (D). The periactive zone area is defined as an area within 68 nm on each side of the peak of the active zone marker (gray shaded areas in B); n as in B. Data are mean ± SEM; *p < 0.05, shown compared to the controlCav2 determined by a two-sided Student’s t-test (D for AP-180) or two-sided Mann–Whitney U tests (C, D for Munc13-1).
Figure 4—figure supplement 3
Additional analyses of en-face synapses after CaV2 ablation in mouse hippocampal neurons.
Quantification of the average integrated intensities (calculated as the object area multiplied by its average fluorescence intensity) of the Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 objects detected in the en-face synapses quantified in Figure 4L–Q. Intensities are normalized to the average signals in controlCav2 per culture; n as in Figure 4P. Data are mean ± SEM; *p < 0.05 determined by Mann–Whitney U tests.
Figure 5 with 3 supplements
Recruitment of endocytic proteins to the periactive zone after active zone disruption in mouse hippocampal neurons.
(A) Schematics of the Rims1, Rims2, Erc1, and Erc2 mutant alleles that constitute the conditional RIM +ELKS quadruple knockout mouse line as described in Tan et al., 2022; Wang et al., 2016. (B–I) Example side-view synapses and average line profiles of Amphiphysin and PSD-95 (B, C), PIPK1γ (D, E), AP-180 (F, G), and Dynamin-1 (H, I). Neurons were stained for a protein of interest (Amphiphysin, PIPK1γ, AP-180, or Dynamin-1; imaged in STED), a postsynaptic marker (PSD-95; imaged in STED), and a synaptic vesicle marker (Synaptophysin or Synapsin; imaged in confocal). An area of interest was positioned perpendicular to the center of the PSD-95 object, and synapses were aligned via the peak fluorescence of PSD-95 in the average line profiles (C, E, G, I). Line profile plots were normalized to the average signal in the controlR+E condition. Dashed lines in C, E, G, and I mark average levels in the controlR+E condition and gray shaded areas represent the active zone and periactive zone area; n in B, C (synapses/cultures): controlR+E 55/3, cQKOR+E 53/3; D, E: controlR+E 55/3, cQKOR+E 54/3; F, G: controlR+E 53/3, cQKOR+E 54/3; H, I: controlR+E 54/3, cQKOR+E 53/3. (J–L) Quantification and statistical analyses of the experiment shown in B–I, including-to-peak distance of PSD-95 and the protein of interest (J), peak intensity in the periactive zone area (K), and peak intensity of PSD-95 (L). The periactive zone area is defined as an area between –136 nm and the peak of PSD-95 (gray shaded areas in C, E, G, I); n as in B–I. (M–R) Example en-face synapses (M–P) and quantification of the number of objects (Q) and distance of these objects to the center of the PSD-95 object (R) of the experiment shown in B–L; n in Q, R: Amphiphysin, controlR+E 22/3, cQKOR+E 23/3; PIPK1γ controlR+E 15/3, cQKOR+E 18/3; AP-180 controlR+E 20/3, cQKOR+E 19/3; Dynamin-1 controlR+E 17/3, cQKOR+E 16/3. Data are mean ± SEM; ***p < 0.001 as determined by two-sided Student’s t-tests (L, Q for Amphiphysin, AP-180, and Dynamin-1; R for PIPK1γ and Dynamin-1) or two-sided Mann–Whitney U tests (J, K, Q for PIPK1γ; R for Amphiphysin and AP-180). For quantification of confocal signals, see Figure 5—figure supplement 1; for assessment of AP-180 using an independent antibody, see Figure 5—figure supplement 2; for additional assessment of en-face synapses, see Figure 5—figure supplement 3.
Figure 5—figure supplement 1
Additional analyses of endocytic proteins after active zone disruption in mouse hippocampal neurons.
(A, B) Example confocal images (A) and quantification of the average intensities (B) of Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 at synapses identified as Synaptophysin or Synapsin puncta. Intensities are normalized to the average signals in controlR+E per culture; n in B (images/cultures): Amphiphysin, controlR+E 15/3, cQKOR+E 16/3; PIPK1γ, 16/3; AP-180, 16/3; Dynamin-1, controlR+E 18/3, cQKOR+E 19/3; PSD-95, controlR+E 65/3, cQKOR+E 67/3; Synaptophysin, controlR+E 47/3, cQKOR+E 48/3; Synapsin, controlR+E 18/3, cQKOR+E 19/3. Data are mean ± SEM; *p < 0.05, ***p < 0.001 determined by two-sided Student’s t-tests for Dynamin-1 and Synapsin or by two-sided Mann–Whitney U tests for Amphiphysin, PIPK1γ, AP-180, PSD-95, and Synaptophysin.
Figure 5—figure supplement 2
Assessment of AP-180 with an alternate antibody after active zone disruption in mouse hippocampal neurons.
(A, B) Example side-view synapses (A) and average line profiles (B) of AP-180 (antibody A246) and PSD-95. Neurons were stained for AP-180 (imaged in STED), PSD-95 (imaged in STED), and the synaptic vesicle marker Synaptophysin (imaged in confocal). A line profile was positioned perpendicular to the center of the PSD-95 object, and synapses were aligned via the peak fluorescence of PSD-95 in the average profiles. Line profiles were normalized to the average signal in controlR+E. Dashed lines mark average levels in the controlR+E condition, and gray shaded areas represent the active zone area; n in b (synapses/cultures): controlR+E 52/3, cQKOR+E 46/3. (C, D) Quantification of the peak-to-peak distance of PSD-95 and AP-180 (C) and of their peak levels in the periactive zone area (D). The periactive zone area is defined as the area –136 nm from the PSD-95 peak toward the presynaptic bouton (gray shaded areas in B); n as in B. Data are mean ± SEM; *p < 0.05, shown compared to the controlCav2 condition determined by a two-sided Student’s t-test (D for AP-180) or two-sided Mann–Whitney U tests (C, D for Munc13-1).
Figure 5—figure supplement 3
Additional analyses of en-face synapses after active zone disruption in mouse hippocampal neurons.
Quantification of the average integrated intensities (calculated as the object area multiplied by its average fluorescence intensity) of the Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 objects detected in the en-face synapses quantified in Figure 5M–R. Intensities are normalized to the average signal in controlR+E per culture, n as in Figure 5Q. Data are mean ± SEM; **p < 0.01 ***p < 0.001 determined by two-sided Mann–Whitney U tests.
Figure 6
Recruitment of endocytic proteins to the periactive zone after disrupting active zone assembly at Drosophila NMJs.
(A) Schematic of the brp69 and brpDef alleles; dashed lines indicate the extent of deletions. (B–E) Example maximum intensity projections of individual boutons of muscle 4 Type 1b terminals from control white and brp69/brpDef larvae (B) and quantification of the average fluorescence intensity per bouton (C), average intensity at the periactive zone mesh (D), and polarization within periactive zone units (E) for Dynamin and Nervous Wreck. Data in C and D are normalized to the average control condition; n in C (NMJs/larvae): brp control 31/3, brp69/Def 26/3; D, E, brp control 31/3, brp69/Def 24/3. Data are mean ± SEM; statistical significance assessed using two-sided Student’s t-tests (C for Nervous Wreck; D for Nervous Wreck; E for Nervous Wreck) or two-sided Mann–Whitney U tests (C for Dynamin; D for Dynamin; E for Dynamin). Images acquired by Airyscan microscopy.
Figure 7 with 2 supplements
Deployment of endocytic proteins after Liprin-α ablation in mouse hippocampal neurons.
(A) Schematics of the Ppfia1, Ppfia2, Ppfia3, and Ppfia4 mutant alleles that constitute the conditional Liprin-α quadruple knockout mouse line as described in Emperador-Melero et al., 2024. (B–I) Example side-view synapses and average line profiles of Amphiphysin and PSD-95 (B, C), PIPK1γ (D, E), AP-180 (F, G), and Dynamin-1 (H, I). Neurons were stained for a protein of interest (Amphiphysin, PIPK1γ, AP-180, or Dynamin-1; imaged in STED), a postsynaptic marker (PSD-95; imaged in STED), and a synaptic vesicle marker (Synaptophysin or Synapsin; imaged in confocal). An area of interest was positioned perpendicular to the center of the PSD-95 object, and synapses were aligned via the peak fluorescence of PSD-95 in the average profiles (C, E, G, I). Line profile plots were normalized to the average signal in the controlL1-L4 condition. Dashed lines in C, E, G, and I mark average levels in the controlL1-L4 condition, and gray shaded areas represent the active zone and periactive zone area; n in B (synapses/cultures), C: controlL1-4 54/3, cQKOL1-4 64/3; D, E: controlL1-4 55/3, cQKOL1-4 50/3; F, G: controlL1-4 59/3, cQKOL1-4 59/3; H, I: controlL1-4 45/3, cQKOL1-4 46/3. (J–L) Quantification and statistical analyses of the experiment shown in B-I, including peak-to-peak distance of PSD-95 and the protein of interest (J), peak intensity in the periactive zone area (K), and peak intensity of PSD-95 (L). The periactive zone area is defined as an area between –136 nm and the peak of PSD-95 (gray shaded areas in C, E, G, I); n as in B-I. (M–R) Example en-face synapses (M–P) and quantification of the number of objects (Q) and distance of these objects to the center of the PSD-95 object (R) of the experiment shown in B-L; n in Q, R: Amphiphysin, controlL1-4 20/3, cQKOL1-4 18/3; PIPK1γ controlL1-4 18/3, cQKOL1-4 16/3; AP-180 controlL1-4 20/3, cQKOL1-4 15/3; Dynamin-1 controlL1-419/3, cQKOL1-4 21/3. Data are mean ± SEM; *p < 0.05, ***p < 0.001 determined by two-sided Student’s t-tests (J for PIPK1γ; K for Amphiphysin; Q for Amphiphysin, AP-180, and Dynamin-1; R for Amphiphysin, AP-180, and Dynamin-1) or two-sided Mann–Whitney U tests (J for Amphiphysin, AP-180, and Dynamin-1; K for PIPK1γ, AP-180, and Dynamin-1; L, Q for PIPK1γ; R for PIPK1γ). For quantification of confocal signals, see Figure 7—figure supplement 1; for additional assessment of en-face synapses, see Figure 7—figure supplement 2.
Figure 7—figure supplement 1
Confocal microscopic analyses of synapses after Liprin-α ablation in mouse hippocampal neurons.
(A, B) Example confocal images (A) and quantification of the average intensities (B) of Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 at synapses identified as Synaptophysin or Synapsin puncta. Intensities are normalized to the average signal in controlL1-4 per culture; n in B (images/cultures): Amphiphysin 26/3, PIPK1γ 16/3, AP-180 20/3, Dynamin-1 18/3, PSD-95 80/3, Synaptophysin 62/3, Synapsin 18/3. Data are mean ± SEM; *p < 0.05 determined by two-sided Student’s t-tests for Synapsin and Dynamin-1 or two-sided Mann–Whitney U tests for Amphiphysin, PIPK1γ, AP-180, PSD-95, and Synaptophysin.
Figure 7—figure supplement 2
Additional analyses of en-face synapses after Liprin-α ablation in mouse hippocampal neurons.
Quantification of the average integrated intensities (calculated as the object area multiplied by its average fluorescence intensity) of the Amphiphysin, PIPK1γ, AP-180, and Dynamin-1 objects detected in the en-face synapses quantified in Figure 7M–R. Intensities are normalized to the average signals in controlL1-4 per culture; n as in Figure 7Q. Data are mean ± SEM; statistical significance was assessed by two-sided Mann–Whitney U tests.
Figure 8
Endocytic proteins are recruited to periactive zones of Drosophila NMJs in Liprin-α mutants.
(A) Schematic of the Liprin-αR60 and Liprin-αF3-ex15 alleles; dashed lines indicate the extent of deletions. (B–G) Example maximum intensity projections of muscle 4 Type 1b terminals from Liprin-αR60/F3-ex15 mutant and white control larvae (B), and quantification of the number of Brp objects per µm2 of NMJ (C) and of their integrated intensity (D). For Brp, Dynamin, Nervous Wreck, EndoA, and Dap160, the average fluorescence intensity per bouton (E), the average intensity at the periactive zone mesh (F), and the polarization within periactive zone units (G) were quantified. Data in D, E, and F are normalized to the average of the control condition; n in C, D, and E–G for Nervous Wreck, Dynamin, and Brp (NMJs/larvae): control 27/5, Liprin-αR60/F3-ex15 26/5; E for Dap160 and EndoA: control 21/6, Liprin-αR60/F3-ex15 19/6; F, G for Dap160 and EndoA: control 20/6, Liprin-αR60/F3-ex15 19/6. Data are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 determined by two-sided Student’s t-tests (E for Dynamin and Nervous Wreck; F, G for Dynamin, EndoA, and Dap160) or two-sided Mann–Whitney U tests (C–E for Brp, EndoA, and Dap160; G for Brp and Nervous Wreck). Images acquired by Airyscan microscopy.
Figure 9
Recruitment of endocytic proteins at Drosophila NMJs in rab3 mutants.
(A) Schematic of the rab3rup allele; red triangle indicates site of 5-bp deletion and frameshift that eliminates the final 35 amino acids (highlighted in red). (B–G) Example maximum intensity projections of muscle 4 Type 1b terminals from rab3rup mutants and white larvae controls (B), and quantification of the number of Brp objects per µm2 of NMJ (C) and of their integrated intensity (D). For Brp, Dynamin, and Nervous Wreck, the average fluorescence intensity per bouton (E), the average intensity at the periactive zone mesh (F), and the polarization within periactive zone units (G) were quantified. Data in D, E, and F are normalized to the average of the control condition; n in C, D (NMJs/larvae): control 22/5, rab3rup 28/6; E: control 25/5, rab3rup 30/6; F, G: control 23/5, rab3rup 23/6. (H, I) Quantification of the levels of Dynamin (H) and of its polarization (I); n in H, I: control Brp+, 23/5, control Brp- 21/6, rab3rup Brp +23/6, rab3rup Brp- 28/6; (J, K) Same as H, I, but for Nervous Wreck; n as in H, I. (L–N) Example maximum intensity projections (L) of muscle 4 Type 1b terminals co-stained for the active-zone marker Brp, the postsynaptic density marker Pak, and the periactive zone marker Fasciclin-II (including the periactive zone segmentation, bottom image), and quantification of the percentage of individual periactive zone segments that contain Brp (M) or are apposed to Pak (N). Arrowheads point at periactive zones lacking Brp. n control 11/3, rab3rup 9/3. Data are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 determined by two-sided Mann–Whitney U tests (C–G, M, N) or by Kruskal–Wallis tests followed by Holm post hoc tests (H–K). In H-K, data are compared to the control Brp +condition. Images acquired by Airyscan microscopy.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Cacna1a conditional knockout | Todorov et al., 2006 | ||
| Genetic reagent (M. musculus) | Cacna1b conditional knockout | Held et al., 2020 | ||
| Genetic reagent (M. musculus) | Cacna1e conditional knockout | Pereverzev et al., 2002 | ||
| Genetic reagent (M. musculus) | Rims1 conditional knockout | Kaeser et al., 2008 | RRID:IMSR_JAX:015832 | |
| Genetic reagent (M. musculus) | Rims2 conditional knockout | Kaeser et al., 2011 | RRID:IMSR_JAX:015833 | |
| Genetic reagent (M. musculus) | Erc1 conditional knockout | Liu et al., 2014 | RRID:IMSR_JAX:015830 | |
| Genetic reagent (M. musculus) | Erc2 conditional knockout | Kaeser et al., 2009 | RRID:IMSR_JAX:015831 | |
| Genetic reagent (M. musculus) | Ppfia1 conditional knockout | Emperador-Melero et al., 2024 | RRID:IMSR_EUMMCR:25506 | |
| Genetic reagent (M. musculus) | Ppfia2 conditional knockout | Emperador-Melero et al., 2021b | RRID:IMSR_HAR:6799 | |
| Genetic reagent (M. musculus) | Ppfia3 constitutive knockout | Wong et al., 2018 | ||
| Genetic reagent (M. musculus) | Ppfia4 conditional knockout | Emperador-Melero et al., 2024 | RRID:IMSR_EUMMCR:3103 | |
| Cell line (Homo sapiens) | HEK 293T cells | ATCC | Cat# CRL-3216; RRID:CVCL_0063 | |
| Recombinant DNA reagent | pFSW HA-Liprin-α3 (plasmid) | Wong et al., 2018 | p526 | |
| Recombinant DNA reagent | pFSW (plasmid) | Nyitrai et al., 2020 | p008 | |
| Recombinant DNA reagent | pFSW GFP Cre (plasmid) | Kaeser et al., 2011 | p009 | |
| Recombinant DNA reagent | pFSW GFP inactive Cre (plasmid) | Kaeser et al., 2011 | p010 | |
| Antibody | anti-Dynamin-1 (Mouse polyclonal) | Milosevic et al., 2011 | IF (1:50) | |
| Antibody | anti-Amphiphysin (Rabbit polyclonal) | Di Paolo et al., 2002 | IF (1:200) | |
| Antibody | anti-PIPK1γ (Rabbit polyclonal) | Wenk et al., 2001 | IF (1:500) | |
| Antibody | anti-AP180 (Mouse monoclonal) | Koo et al., 2015 | IF (1:500) | |
| Antibody | anti-AP180 (Rabbit polyclonal) | SySy | RRID:AB_887691 | IF (1:500) |
| Antibody | anti-Munc13-1 (Rabbit polyclonal) | SySy | RRID:AB_887733 | IF (1:500) |
| Antibody | anti-Synaptophysin (Mouse monoclonal) | SySy | RRID:AB_887824 | IF (1:500) |
| Antibody | anti-Bassoon (Mouse monoclonal) | Enzo | RRID:AB_11181058 | IF (1:500) |
| Antibody | anti-PSD-95 (Mouse monoclonal) | Neuromab | RRID:AB_10698024 | IF (1:500) |
| Antibody | anti-PSD-95 (Guinea pig monoclonal) | SySy | RRID:AB_2619800 | IF (1:500) |
| Antibody | anti-Gephyrin (Mouse monoclonal) | SySy | RRID:AB_2232546 | IF (1:500) |
| Antibody | anti-Synapsin-1 (Rabbit polyclonal) | SySy | RRID:AB_2200097 | IF (1:500) |
| Antibody | anti-Nervous Wreck 970 (Rabbit polyclonal) | Coyle et al., 2004 | RRID:AB_2567353 | IF (1:1000) |
| Antibody | anti-Dynamin (Guinea Pig polyclonal) | Provided by Dion Dickman | IF (1:1000) | |
| Antibody | anti-Brp nc82 (Mouse monoclonal) | Developmental Studies Hybridoma Bank (DSHB) | RRID:AB_2314866 | IF (1:1000) |
| Antibody | anti-Pak (Rabbit polyclonal) | Harden et al., 1996 | IF (1:1000) | |
| Antibody | anti-Synapsin (Mouse monoclonal) | Developmental Studies Hybridoma Bank (DSHB) | RRID:AB_528479 | IF (1:100) |
| Antibody | anti-EndoA (Rabbit polyclonal) | Provided by Dion Dickman | IF (1:1000) | |
| Antibody | anti-Dap160 (Guinea Pig polyclonal) | This study | IF (1:1000) | |
| Chemical compound, drug | ω-Agatoxin IVA | Alomone labs | Cat# STA-500 | 200 nM |
| Chemical compound, drug | ω-Conotoxin GVIA | Alomone labs | Cat# C-300 | 250 nM |
| Chemical compound, drug | Tetrodotoxin | Tocris Bioscience | Cat# 1078 | 1 µM |
| Genetic reagent (Drosophila melanogaster) | GMR94G06-GAL4 | Bloomington Drosophila Stock Center (BDSC); Jenett et al., 2012 | RRID:BDSC_40701 | |
| Genetic reagent (Drosophila melanogaster) | UAS-TeTxLC; UAS-TeNT | BDSC | RRID:BDSC_28838 | |
| Genetic reagent (Drosophila melanogaster) | brpDf/CyOGFP | Akbergenova et al., 2018 | ||
| Genetic reagent (Drosophila melanogaster) | brp69 | Kittel et al., 2006 | ||
| Genetic reagent (Drosophila melanogaster) | rab3rup | Graf et al., 2009 | RRID:BDSC_78045 | |
| Genetic reagent (Drosophila melanogaster) | liprinR60 | BDSC; Kaufmann et al., 2002 | RRID:BDSC_8561 | |
| Genetic reagent (Drosophila melanogaster) | liprinF3ex15 | BDSC; Kaufmann et al., 2002 | RRID:BDSC_8563 |
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Deployment of endocytic machinery to periactive zones of nerve terminals is independent of active zone assembly and evoked release
eLife 14:RP107276.
https://doi.org/10.7554/eLife.107276.3
