Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A ) Schematic diagram represents the protein structural elements in proline rich region (PRR) of the Dyn1 splice variants. Dyn1xA and xB share identical amino acid sequence up to Pro844. Blue color shows previously reported Endophilin A binding region. Green color shows 20 amino acid extension of Dyn1xA. Light green color shows calcineurin binding sites specific to Dyn1xB. Ser845 is the splice site boundary. ( B ) Synaptosomal lysates were incubated with GST-Dyn1-PRR (either xA or xB) coupled to GSH-sepharose beads. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue. The bands marked as Endophilin A were subjected to LC–MS/MS analysis. Samples are from duplicate experiments. Note that the two GST-tagged PRR baits are overloaded and are of different sizes (left vs right panels). ( C ) Bound proteins from pull-down experiments with GST-Dyn1-PRR (either xA or xB) were subjected to Western blot analysis with antibodies against endophilin A, amphiphysin 1, each being run in duplicate experiments. Results are representative of at least two independent experiments. ( D – F ) Schematic diagram represents truncated PRR used in pull-down experiments. Dyn1-751-798 contains the previously reported Endophilin A binding region (blue). Dyn1xA-794-864 and Dyn1xB-794-851 contain Glycine 801 (magenta) which has inhibitory role for Endophilin A binding. Corresponding synaptosomal lysates were incubated with truncation constructs ( D ) coupled to GSH-sepharose beads. Bound proteins were subjected to Western blot analysis with antibodies against Endophilin A and Amphiphysin 1. All conditions are performed on the same blot. Results shown are in duplicate samples from one of at least 3 independent experiments. ( G ) ELISA assays used to determine the binding affinity, demonstrated that the C-terminus Dyn1xA-806-864 is has the major binding site for Endophilin A. His-tagged mouse Endophilin-SH3 domain was coated onto a 96-well plate and examined for the ability to bind to increasing concentrations of a variety of GST-tagged Dyn1-PRR peptides by an ELISA assay. The binding affinity (Kd) for GST-tagged each dynI-PRD construct was calculated based on two experiments, each containing four replicates. Mean ± SD is shown. .

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Sequencing, Binding Assay, Incubation, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Construct, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A ) Rat brain synaptosome lysates were incubated with GST-Dyn1-PRR (either xA or xB) coupled to GSH-sepharose beads. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue. The protein band at 40 kDa from both GST-Dyn1-PRR (either xA or xB) were individually cut and digested using Trypsin/LysC and analyzed using a targeted LC–MS/MS SRM analyses. The total amount of each Endophilin protein bound to each Dyn1-PRR was calculated by summing the total peak area for each Q1/Q3 transition to provide the total peak area. The percentage of the total peak area for each protein was then calculated. Endophilin A1 was the predominant protein with a level of Endophilin A3 at 11-fold lower levels, while A2 levels were more than 250-fold lower than A1. ( B ) List of unique Endophilin isoform-specific peptides used for SRM assay (rat sequences: Endophilin-A1_sp| O35179 |SH3G2, Endophilin-A2-sp| O35964 |SH3G1, Endophilin-A3-sp| O35180 |SH3G3).

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Incubation, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: Partial 15 N-HSQC spectra are shown for 0.27 mM 15 N-DynxA 746-864 alone (red) and for the same protein following the addition of 2.6 molar equivalents of unlabeled Endophilin A1 SH3 domain. Assignments are shown for DynxA alone. Note that some signals for which assignment locations are marked are not visible in this spectrum but were visible in other spectra recorded for longer times. The bottom panels show cropped portions of R846 and F862 from the top panel. Arrows indicate peak shifting during the titration processes.

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Titration

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A , B ) Summary of backbone amide chemical shift perturbations (Δδ in ppm) following titration of Endophilin A1 SH3 into a solution of 15 N-labeled Dyn1xA 751-798 ( A ) or Dyn1xA 806-864 (in B ). ( C ) Summary of the backbone amide chemical shift perturbations (Δδ in ppm) following titration of Endophilin A1 SH3 into a solution of 15 N-labeled GST-tagged Dyn1xA-PRR. PxxP motifs are outlined as green boxes. Phosphobox-1 is outlined as a blue box. .

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Titration, Labeling

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A ) Amino acid sequence of Dyn1xA 806-864 . The amino acids for site-mutagenesis were underlined, and their positions marked with the number. The splice site after Ser845 in Dyn1xA-PRR is indicated with a red line. ( B – E ) Samples from pull-down experiments from brain lysates with the indicated GST-tagged Dyn1xA 806-864 or Dyn1xA-PRR and their specific point mutants, were run on gels, blotted and probed with antibodies to Endophilin A or Amphiphysin 1. Results shown are in duplicate samples from one of at least 3 independent experiments. ( F ) The binding of Endophilin A, Amphiphysin 1 and Syndapin 1 to Dyn1xA-PRR (wild type) or R838A mutant quantified from Western blots in ( E ). n = 14 (6 experiments with 2–4 replicates in each). Median and 95% confidential intervals are shown. Kruskal–Wallis with Dunn’s multiple comparisons test was applied (** p < 0.001). ( G ) The binding of Endophilin A, Amphiphysin 1 and Syndapin 1 to Dyn1xA-PRR (wild type) or R846A mutant quantified from Western blots in ( E ). n = 14 (6 experiments with 2–4 replicates in each). Median and 95% confidential intervals are shown. Kruskal–Wallis with Dunn’s multiple comparisons test was applied (* p < 0.05). ( H ) SEC-MALS profiles for Dyn1xA alone (in green), Endophilin A SH3 alone (in red) and the complex of the two (in black) are plotted. The x-axis shows retention time. The left axis is the corresponding UV absorbance (280 nm) signals in solid lines, and the right axis shows the molar mass of each peak in crosses. The molecular weight of the complex was determined and tabulated in comparison with the predicted molecular weight. x represent individual data points. ( I ) SEC-MALS profiles for a high concentration of Dyn1xA-PRR/Endophilin A SH3 complex (0.5 mg) (in dark blue) and a low concentration of Dyn1xA-PRR/endophilin A SH3 complex (0.167 mg) (in blue). The x-axis shows retention time. The left axis is the corresponding UV absorbance (280 nm) signals in solid lines, and the right axis shows the molar mass of each peak in crosses. The molecular weight of the complex was determined and tabulated in the table. x represent individual data points. ( J ) A schematic diagram of phosphobox-1 and -2 in Dyn1xA-PRR each containing dual phosphorylated serines. ( K ) Phosphomimetic mutants were made in the phosphoboxes. Samples from pull-down experiments with GST tagged PRD and both of its phosphomimetic mutants, S774/778E and S851/857E, GST-Dyn1xA 751-798 and Dyn1xA 751-798 S774/778E, GST- Dyn1xA 794-864 and Dyn1xA 794-864 S774/778E, were run on gels, blotted, and probed with antibodies to Endophilin A and Amphiphysin 1. Results are shown from one of three independent experiments. ( L , M ) The amount of Endophilin A and Amphiphysin 1 bound to full-length Dyn1xA-PRR and its phosphomimetic mutants were quantified by densitometric analysis of the Western blots such as in ( K ). n = 8. All data were expressed as a percent of Dyn1xA-PRR Mean ± SEM. .

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Sequencing, Mutagenesis, Binding Assay, Western Blot, Molecular Weight, Comparison, Concentration Assay

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A ) The top image shows an axon containing multiple boutons. Signals show overexpression of GFP-tagged Dyn1xA (Dyn1xA) and mCherry-tagged Endophilin A1 (EndoA1). The bottom images show magnifications of four boutons in the top image. Red hot LUT images on the right side of Dyn1xA and EndoA1 images are enhanced contrast images. Outer and inner contour 50% and 70% of local maxima of the Dyn1xA, respectively. Black circles represent local maxima of Endophilin A1. In these boutons, there are multiple EndophilinA1 puncta in each bouton. ( B ) The top image shows an axon containing multiple boutons. Signals show overexpression of mCherry-tagged Dyn1xA (Dyn1xA) and GFP-tagged Endophilin A2 (EndoA2). The bottom images show magnifications of four boutons in the top image. Red hot LUT images on the right side of Dyn1xA and EndoA2 images are enhanced contrast images. Outer and inner contour 50% and 70% of local maxima of the Dyn1xA, respectively. Black circles represent local maxima of Endophilin A2. In these boutons, there are multiple EndophilinA2 puncta in each bouton. ( C ) STED micrographs of the same synapses as in Fig. with an active zone marker Bassoon (magenta) visualized by antibody staining of GFP-tagged Dyn1xA, Dyn1xA S851D/857D or Dyn1xA R846A (green). Local maxima of Dyn1xA, Dyn1xA S851D/857D or Dyn1xA R846A signals and minimum distance to the active zone boundary are overlayed.

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Over Expression, Marker, Staining

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: ( A , B ) Example STED micrographs showing overexpression of GFP-tagged Dyn1xA (Dyn1xA) and mCherry-tagged Endophilin A1 (EndoA1) ( A ), mCherry-tagged Dyn1xA and GFP-tagged Endophilin A2 (EndoA2) ( B ). Green dashed line in Endophilin A1 or A2 images indicates boundary of Dyn1xA clusters defined by MATLAB script (see Methods). White dashed line in merged images indicate neuron shape based on background fluorescence. ( C ) Cumulative plot representing distance of Endophilin A1 or A2 puncta from the Dyn1xA boundary. Negative values indicate local maxima of Endophilin puncta are inside the boundary of Dyn1xA puncta and positive values indicate outside. ( D ) Fraction of Dyn1xA puncta contains Endophilin A1 or A2 within the boundary. Data information: data are presented as mean ± SEM. ( E ) Confocal micrographs (top panel) showing overexpression of GFP-tagged Dyn1xA, Dyn1xA S851D/857D or Dyn1xA R846A. Bottom panels show STED micrographs of the same synapses with an active zone marker Bassoon visualized by antibody. False-colored images show the relative fluorescence intensity of Dyn1xA or mutants. White thick dashed lines within false-colored STED images indicate the boundary of active zone based on the MATLAB analysis of Bassoon signals. Data information: data are presented as median with 95% confidence interval. ( F ) The distribution of Dyn1xA, Dyn1xA S851/857D and R846A relative to the active zone edge. Negative values indicate local maxima of Dyn1xA or mutants puncta are inside the active zone, and positive values indicate outside. Data information: * P < 0.05 (Kolmogorov–Smirnov test). ( G ) Cumulative plots of ( F ). More than 150 synapses are examined in each condition. n > 4 coverslips from 2 independent cultures. .

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Over Expression, Fluorescence, Marker

Journal: The EMBO Journal

Article Title: Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis

doi: 10.1038/s44318-024-00145-x

Figure Lengend Snippet: A splice variant of Dynamin 1, Dyn1xA, but not other isoforms/variants, can mediate ultrafast endocytosis. ( A ) Dyn1xA has 20 amino acid extension, containing a newly identified high affinity Endophilin A1 binding site. Three amino acids, R846 at the splice site boundary, S851 and S857, act as long-distance elements to enhance affinity of proline rich motifs (PRM) of Dyn1xA to EndophilinA1-SH3 domain. ( B ) At a resting state, Dyn1xA accumulates at the endocytic zone with SH3 containing BAR protein Syndapin 1 (Imoto et al, ) and Endophilin A1/2. When phosphobox-1 (Syndapin 1 binding) and phosphobox-2 (Endophilin A1/2 binding, around S851/S857) are phosphorylated, Dyn1xA molecules are diffuse within the cytoplasm. Only the dephosphorylated fraction of Dyn1xA molecules can interact with these BAR domain proteins and localize to the endocytic zone. Loss of interactions (i.e., by Dyn1xA-R846A or -S851/857D mutations) disrupts their pre-accumulation at the endocytic zone. Consequently, ultrafast endocytosis fails or slows down.

Article Snippet: Antibodies to Endophilin, Amphiphysin I, and Dynamin IIwere from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies to GFP were from Life Technology and secondary antibodies were all from Dako (Denmark).

Techniques: Variant Assay, Binding Assay