Figure 4.

Endophilin A1 binding to intersectin 1 regulates clathrin uncoating

1. Endophilin A1 binding intersectin 1-SH3B binds via a non-canonical interface on its SH3 domain. GST-fused full-length (FL) endophilin A1 wild-type (WT), a proline-rich peptide binding defective mutant (PxxEY->AxxKA), or a mutant within the intersectin 1-SH3B binding interface (E329K, S336K) were incubated with purified recombinant intersectin 1-SH3B and analyzed by SDS–PAGE and staining with Coomassie blue.
2. Mutant endophilin A1 fails to bind to intersectin 1 while retaining association with proline-rich ligands. (B) GST or GST-fused full-length (FL) endophilin A1 wild-type (WT), a proline-rich peptide binding defective mutant (PxxEY->AxxKA), or a mutant within the intersectin 1-SH3B binding interface (E329K, S336K) were incubated with RBE. Samples were analyzed by immunoblotting for synaptojanin 1 (SJ1), dynamin 1 (Dyn1), or clathrin heavy chain (HC). (C) GST or GST-endophilin A1-SH3 wild-type (WT) or mutant (E329K, S336K) were incubated with RBE. Samples were analyzed by immunoblotting for intersectin 1 (ITSN1), vesicular glutamate transporter 1 (VGLUT1), dynamin 1 (Dyn1), or actin as a negative control.
3. Endophilin A1-mRFP WT and mutant (E329K, S336K) partition equally between membrane and soluble cytosolic fractions of HEK293 cells. Samples were immunoblotted for endophilin A1 (EndoA1), transferrin receptor (TfR), or actin.
4. Endophilin A1 binding to intersectin 1 regulates clathrin uncoating. Quantification of clathrin clustering in cortical neurons (DIV 14–22) from WT or endophilin A1-3 TKO mice re-expressing endophilin A1 WT or mutant (E329K, S336K). Clustering was fully rescued by endophilin A1-mRFP WT, but only to a minor degree by mutant endophilin A1 (E329K, S336K); y-axis, fold increase of fluorescence puncta in mutant synapses normalized to WT. *P < 0.05, **P < 0.01, ***P < 0.001, t-test.
5. Equal expression of endophilin 1 A1-mRFP wild-type (WT) or mutant (E329K, S336K) in primary cortical neurons (DIV 14–22; 68 random images from n = 4 independent experiments).
6. Intersectin 1 regulates endophilin A1 targeting to sites of clathrin-mediated endocytosis in neurons. Colocalization between endogenous endophilin A1 and AP-2 in primary hippocampal neurons (DIV 14) from wild-type (WT) or intersectin 1 knockout (KO) mice assessed by Pearson's correlation (four independent experiments; 111 for WT and 110 random images for KO). Data represent mean ± SEM. ***P < 0.0001, two-tailed unpaired t-test.