Fig 2: The clathrin adaptor CALM is enriched at postsynaptic endocytic zones where AMPAR endocytosis occurs.(A) During LTD induction, CP-AMPARs are recruited into synaptic regions, whereas CI-AMPARs are internalized via CME by binding to AP-2. Within a few minutes of LTD progression, the increased Ca2+ concentration activates phosphatases inducing GluA1 dephosphorylation and subsequent CP-AMPAR removal by a still unknown endocytic mechanism. (B and C) Tryptic digest of synaptosomes reveals postsynaptic localization of CALM. (B) Synaptosomes were left untreated or incubated with trypsin and analyzed by immunoblotting. (C) For quantification, trypsin-treated samples were normalized to untreated controls. (D and E) Three-channel time-gated STED confirms predominant postsynaptic localization of CALM. (D) Representative image of wild-type (WT) hippocampal neurons immunolabeled with antibodies against the presynaptic marker Bassoon, the postsynaptic marker Homer 1, and CALM (scale bar, 500 nm). Yellow line indicates position of the normalized fluorescent intensity depicted in (E). (F) The majority of CALM resides outside of the PSD. WT mouse brain homogenates were subjected to subcellular fractionation, and equal protein amounts of total homogenate (T), cytosolic fraction (S2), synaptosomes (P2), synaptosomal membranes (P3), synaptic plasma membranes (SPMs), and PSD were compared by immunoblotting with the indicated markers. Statistics Table 1. ns, not significant.

Fig 3: Loss of neuronal CALM selectively impairs the endocytosis of GluA1 homomers.(A and B) GluA1 is missorted upon CALM KO. Surface/total ratios of SEP-GluA1 (A) or SEP-GluA2 (B) expressed in WT or KOCAG neurons. (C) Rescue of increased GluA1 surface level in KOCAG neurons by CALM reexpression. Surface/total protein ratios of SEP-GluA1 in neurons transduced with pAAV-iRFP or pAAV-CALM-WT. (D and E) CALM deletion causes increased surface levels of endogenous GluA1. After surface biotinylation of neurons, total and biotinylated proteins were analyzed by immunoblotting (D). Surface/total ratios normalized to WT (E). (F) CALM KO causes the accumulation of GluA1 homomers. Surface/total protein ratios of SEP-tagged GluA1 or GluA2 in WT or KOCAG neurons cotransfected with HA-GluA1 or HA-GluA2 to unravel differences between homomers and heteromers. (G to I) Surface accumulation of GluA1 upon CALM loss not due to altered exocytosis or lateral diffusion. WT and CALM KOCAG neurons coexpressing SEP-GluA1 (green) and dendritic mCherry-spinophilin (magenta) (G) were photobleached at synaptic and extrasynaptic regions (H) (scale bar, 1 µm). Percent fluorescence recovery at 6 to 8 min after beaching (I). (J to M) GluA1 endocytosis is impaired in the absence of CALM. WT and CALM KOCAG neurons live-labeled using antibodies recognizing GluA1 (J) or GluA2 (L) were fixed and immunostained in the absence/presence of detergent to label the surface or internal pool (images: scale bar, 5 µm). GluA1 (K) or GluA2 (M) uptake normalized to WT. Statistics Table 1.

Fig 4: GluA1 endocytosis is driven by CALM-mediated membrane remodeling and independent of clathrin.(A and B) CALM-mCherry is present during GluA1 endocytosis events detected with ppH assay at pH 5.5 (A). (B) Average fluorescence intensity aligned to vesicle detection time (frame 0; gray areas, 95% confidence interval of randomized data). (C) Coimmunoprecipitation of endogenous GluA1-CALM complexes from synaptosomal membranes using GluA1- or CALM-specific antibodies. (D) CALM-ANTH domain binds cytosolic GluA1–C terminus. GST–GluA1–C terminus or GST was incubated with His-tagged WT-CALM-ANTH or cargo binding–deficient mutant (?cargo). Analysis by immunoblotting. (E) Surface levels of GluA1-chimera containing C-terminal domain of GluA2 are unaffected by CALM loss. Neuronal surface/total protein ratios of SEP-GluA1-chimera assessed by acid-base quenching. (F) CALM mutants of (G). (G) CALM’s function in GluA1 endocytosis depends on its PI(4,5)P2 binding and membrane curvature induction but is clathrin independent. SEP-GluA1 surface/total levels in neurons coexpressing iRFP (control), WT-CALM, or CALM-mutant deficient in PI(4,5)P2 binding, membrane insertion (?H0), GluA1 binding (?cargo), or clathrin binding. (H) shRNA-mediated clathrin depletion. Lysates of WT neurons expressing scrambled or anti-CHC shRNA were probed by immunoblotting (clathrin protein levels normalized to SNAP-25). (I and J) Endocytosis of Alexa Fluor 647–transferrin is impaired in clathrin-depleted cells (images: scale bar, 20 µm; nuclear RFP, transduced cells). Values normalized to control cells. (K) Increased transferrin receptor surface pool in clathrin-depleted neurons expressing pHuji-TrfR. (L) Unaltered GluA1 surface pool in clathrin-depleted neurons expressing SEP-GluA1. Statistics Table 1. a.u., arbitrary units.

Fig 5: Newly exocytosed proteins co-cluster with endocytic proteins in the periactive zone.(a) 3D-Structured Illumination Microscopy (3D-SIM) analysis of Bassoon (Bass), AP180 and clathrin heavy chain (CHC) in stimulated (40 APs, 20 Hz) or non-stimulated hippocampal neurons. Scale bar, 1 µm; zoom, 500 nm. (b) Schematic for immunolabeling of the newly exocytosed SV protein pool (NEP). (c–e) SD-dSTORM imaging of newly exocytosed SV proteins (stimulation: 40 AP, 20 Hz) in hippocampal neurons expressing Syb2-c-myc. (c) 2-colour SD-dSTORM analysis of NEP and AP180, overlaid with wide-field image of Bassoon. Scale bar, 1 µm; zoom, 100 nm. (d) Ripley's L(r)-r function shows sub-synaptic clustering of NEP and of AP180, indicated by positive L(r)-r values with a maximum at radii of about 100 nm (NEP) and 200 nm (AP180). Mean±s.e.m.; n=31 synapses. Dotted lines represent upper envelopes of complete spatial randomness (CSR). (e) Radial intensity profiles of NEP and AP180 signals centred on the maxima of sub-synaptic NEP clusters show colocalization of both proteins (mean±s.e.m.; n=47 synapses). (f) k-Nearest neighbour analysis (k=10) of NEP and AP180 showing a maximum at 25 nm. The red line represents results after toroidal shift of one channel. Values above the red line indicate colocalization of NEP and AP180 (mean±s.e.m.; n=31 synapses).