PMC

Microinjection of CLAP antibodies shifts the proportion of coated pits to early stages and increases their size. (A and B) Shallow coated pits from control (A) and CLAP antibody-injected (B). (C and D) Examples of nonconstricted (bucket-shaped) coated pits from control (C) and CLAP antibody-injected axons (D). (E) Flat clathrin-coated patch on the presynaptic membrane (arrows) from an axon microinjected with CLAP antibodies. A coated pit is also present to the left. (Scale bars: 100 nm.) (F) Quantitative analysis showing representation of different stages of coated pits in synapses microinjected with CLAP antibodies as compared with noninjected controls (n = 181 coated pits for CLAP antibody-injected axons, n = 109 coated pits for control synapses; P < 0.001 with χ² test). (G) Length of the invaginated part of shallow coated pits (P < 0.001, Student's t test) and nonconstricted coated pits (P < 0.001, Student's t test; n = 15 coated pits for each condition).

Plot of Robinson-Foulds (RF) distance between dendrograms from CLAP and ClustalW with respect to different sizes of proteins sequences.

Characterization of a lamprey epsin 1 ortholog. (A) Domain structure of lamprey epsin. Identity to human epsin 1 for each region is indicated in percentage. Antibodies were raised to residues 42–207 (ENTH) and 262–496 (CLAP) (black lines). (B) Liposome sedimentation assay with wild-type ENTH domain (with and without liposomes) and mutant ENTH domain (with and without liposomes), stained with Coomassie blue. (C) GST-CLAP pull-down with lamprey brain extract. Clathrin and AP2 were detected with clathrin heavy chain and α-adaptin antibodies, buffer, or extract with GST alone. (D) Protein extracts from lamprey brain analyzed by SDS/PAGE and Western blot using ENTH and CLAP antibodies. (E) (Upper) Immunoprecipitation of lamprey brain extract with ENTH and CLAP antibodies. Also shown are control incubations with buffer alone and with control IgGs (2.5% of the pellet was loaded). (Lower) Shown is the remaining signal in the unbound material, control IgG, and 1% of the starting material. (F) Western blot analysis of GST-CLAP pull-downs showing selective inhibition of the binding of clathrin and AP2 by CLAP antibodies but not by control IgGs. (G) Pull-down with the GST-β appendage domain of AP2 from lamprey brain extract. Coomassie staining (Left) and silver staining (Right) are shown. (H) Western blot analysis of bound material vs. start material with AP180 and epsin CLAP antibodies (at locations indicated in G). (I) Effects of epsin CLAP and epsin ENTH antibodies on epsin binding (from lamprey brain extract) to the GST-β appendage domain of AP2. Note the reduced binding of epsin induced by the CLAP antibodies.

The period difference as a function of clap timing t _j − t _i at the current clap instance, measuring the response of each individual in clap timing at the next step caused by differences in timing with the partner at the current clap instance. The solid line corresponds to experimental data (with standard error bars), and the dotted line to simulations of Model I.