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 three days starting DIV3. For acute depolarization (“KCl”), neurons were stimulated with 50 mM KCl for 30 seconds 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. Grey dotted lines in c, e, g and i mark average levels in the untreated condition and grey bars 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 of the 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 (grey shaded areas in c, e, g and 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-I, 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, and q), or by one-way ANOVA followed by a Tukey-Kramer post hoc test (j for Dynamin-1). For quantification of confocal images, see Fig. 1 - figure supplement 1; for a workflow of STED analyses, see Fig. 1 - figure supplement 2; for assessment of AP-180 using and independent antibody, see Fig. 1 - figure supplement 3, for additional analyses of en-face synapses, see Fig. 1 – figure supplement 4.

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 three minutes (“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 (b), and quantification of the number of Brp objects per µm2 of NMJ (c) and 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 g are normalized to the average of the control condition; n in c, d (NMJs/larvae): control 28/5, TeNT 27/6; e: control 28/5, TeNT 28/6; f, g: control 28/5, TeNT 22/6.

(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: no stim 26/14, 40 Hz 25/14; l, m: no stim 26/14, 40 Hz 23/14.

Data are mean ± SEM; *p < 0.05 determined by two-sided Student’s t-tests (e for Nervous Wreck; g for Brp and Nervous Wreck; i; j; k; m) or two-sided Mann-Whitney U tests (c; d; e for Brp and Dynamin; f; g for Dynamin; l).

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 profiles plots were normalized to the average signal in the controlCav2 condition. Turquoise dotted lines in c, e, g and i mark average levels in the controlCav2 condition and grey bars 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 of the 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 (grey shaded areas in c, e, g and 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-I, 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, 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 Fig. 3 - figure supplement 1; for assessment of AP-180 using and independent antibody, see Fig. 3 - figure supplement 2; for additional assessment of en-face synapses, see Fig. 3 – figure supplement 3.

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.

Orange dotted lines in c, e, g and i mark average levels in the controlR+E condition and grey bars 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 of the 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 (grey shaded areas in c, e, g and 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-I, 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 Fig. 4 - figure supplement 1; for assessment of AP-180 using and independent antibody, see Fig. 4- figure supplement 2; for additional assessment of en-face synapses, see Fig. 4 – figure supplement 3.

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/brpDeflarvae (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).

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-l) 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. Orange dotted lines in c, e, g and i mark average levels in the controlL1-L4 condition and grey bars 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 of the 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 (grey shaded areas in c, e, g and 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-I, 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 Fig. 6- figure supplement 1; for additional assessment of en-face synapses, see Fig. 6 – figure supplement 2.

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 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-g (NMJs/larvae): control 27/5, Liprin-αR60/F3-ex15 26/5.

Data are mean ± SEM; * p<0.05 ** p<0.01 *** p<0.001 determined two-sided Student’s t-tests (e for Dynamin and Nervous Wreck; f, g for Dynamin) or two-sided Mann-Whitney U tests (c, d, e for Brp; g for Brp and Nervous Wreck).

Recruitment of endocytic proteins at Drosophila NMJs of 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 (k) and of its polarization (l); n in k, l: 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); 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, d, e, f, 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.

Confocal microscopic analyses of synapses after chronic silencing or acute depolarization of mouse hippocampal neurons

(a, b) Example confocal images (a) and quantification (b) of the average intensities 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 a Holm post hoc tests for Amphiphysin, PIPK1γ, AP-180, Dynamin-1, Synaptophysin and Munc13-1.

Workflows for STED analyses in mouse hippocampal neurons and for confocal analyses at Drosophila neuromuscular junctions

(a-e) 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), Amphiphysin (imaged in STED) and Synaptophysin (imaged in confocal). Synapse selection and placement of an area of interest (white transparent rectangle with line profile direction indicated by black arrow) perpendicular to the marker (Bassoon, in this example) is done by an experimenter blind for the protein of interest (Amphiphysin, in this example). Next, the protein of interest channel is activated (b), and the profile is generated (c). Then, the average from many synapses is calculated and plotted aligned to the peak of the marker protein (d). The distance of the peak to the peak of the marker protein and the maximum intensity within the region corresponding to the periactive zone are also calculated and plotted (e). The example corresponds to the untreated condition in Fig. 1b+c.

(f-i) Workflow for the analyses of en-face synapses of mouse hippocampal neurons, showing an example synapse immunostained for Bassoon (imaged in STED), Amphiphysin (imaged in STED) and Synaptophysin (imaged in confocal). First, synapse selection is done by an experimenter blind for the protein of interest (f). Next, the channel of the protein of interest is activated (g), and objects containing endocytic proteins and the marker are identified in the respective channels using an algorithm (h). Finally, the number, integrated intensites, and the distances of the objects to the center of the marker are quantified (i). The example corresponds to the untreated condition in Fig. 1l.

(j-n) Workflow for analyses of Drosophila neuromuscular junctions. Terminals are analyzed both in 3D (k) and in 2D half-maximum intensity projections (j, l-n). 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 (k). Next, the periactive zone levels and degree of polarization are analyzed in 2D half-maximum intensity projections. 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. The resulting ROIs are used to measure average intensities within the mesh and the core. Levels are plotted as normalized intensities in the mesh, and the mesh:core ratios are calculated to determine polarization (n). Ratios greater than zero indicate enrichment of the protein at the periactive zone, while those less than zero indicate de-enrichment.

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 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. Grey dotted lines mark average levels in the untreated condition and grey bars 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 (grey shaded areas in b)(Held et al., 2020), 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.

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 Fig. 1l-q. Intensities are normalized to the average signals in the untreated conditions per culture; n as in Fig. 1p.

Data are mean ± SEM; **p < 0.05, ***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.

Confocal microscopic analyses of synapses after CaV2 ablation in mouse hippocampal neurons

(a, b) Example confocal images (a) and quantification (b) of the average intensities 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 a 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.

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 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 controlCav2 condition. Green dotted lines mark average levels in the controlCav2 and grey bars 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 (grey 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).

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 Fig. 3l-q. Intensities are normalized to the average signals in controlCav2 per culture; n as in Fig. 3p.

Data are mean ± SEM; *p < 0.05 determined by Mann-Whitney U tests.

Additional analyses of endocytic proteins after active zone disruption in mouse hippocampal neurons

(a, b) Example confocal images (a) and quantification (b) of the average intensities 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.

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 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. Green dotted lines mark average levels in the controlR+E condition and grey bars represent the active zone area, n in b (synapses/cultures): controlR+E, 52/3 cultures; 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 towards the presynaptic bouton (grey 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).

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 Fig. 4m-r. Intensities are normalized to the average signal in controlR+E per culture, n as in Fig. 4q.

Data are mean ± SEM; **p < 0.01 ***p < 0.001 determined by two-sided Mann-Whitney U tests.

Confocal microscopic analyses of synapses after Liprin-α ablation in mouse hippocampal neurons

(a, b) Example confocal images (a) and quantification (b) of the average intensities 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.

Additional analyses of en-face synapses after Liprin-α ablation in mouse hippocampal neurons

(c) 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 Fig. 6m-r. Intensities are normalized to the average signals in controlL1-4 per culture; n as in Fig. 6q.

Data are mean ± SEM; statistical significance was assessed by two-sided Mann-Whitney U tests.