BAR scaffolds drive membrane fission by crowding disordered domains

Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak

The pCAGEN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1-256), tagged at the C-terminus with mCherry, was a gift from Tobias Meyer (Addgene plasmid # 85130). Full-length human amphiphysin (residues 1-695) was cloned into the pCAGEN vector, in frame with mCherry at the C-terminus, by first excising the N-BAR domain from the template using EcoRI and AgeI restriction sites, and then ligating in full-length amphiphysin using the same EcoRI and AgeI restriction sites. The N-BAR domain of human amphiphysin fused to the C-terminal domain of mouse neurofilament-M (N-BAR-NfM CTD, residues 411-848 of mouse neurofilament-M) was cloned by ligating neurofilament-M CTD into the existing N-BAR-mCherry pCAGEN template, between N-BAR and mCherry, using a single AgeI restriction site. The resulting plasmid contained a GPV linker between N-BAR and neurofilament-M CTD and a GPVAT linker between neurofilament-M CTD and mCherry. All plasmids were confirmed by DNA sequencing.