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

A plasmid for expression of BFP-tagged clathrin light chain (BFP-CLC) was generated by replacing the mCherry domain of mCherry-CLC, a gift from Dr. Tom Kirchhausen (Addgene #53972). The mCherry fluorophore was removed and replaced with tagBFP, a gift from Dr. Franck Perez (Addgene #65257). mCherry was excised from the mCherry-CLC plasmid using AgeI and XhoI restriction enzymes. TagBFP was amplified from the li-Str_ManII-SBP-tagBFP plasmid using PCR primers which introduced AgeI and XhoI restriction sites. The resulting tagBFP sequence was digested and ligated onto the CLC backbone to generate BFP-CLC with a linker sequence of HKGRPTR. The CLC-BFP construct was then excised using AgeI and EcoRI restriction sites and ligated into a pLJM1 backbone obtained from Addgene as a gift from Dr. David Sabatini (Addgene #19319). Once subcloned into this viral transfer plasmid, lentiviruses were generated by transfecting the BFP-CLC construct with the envelope plasmid VSVG (a gift from Dr. Jennifer Lippincott-Schwartz, Addgene #11912) and packaging plasmid pCMV-dR8.91 (a gift from Dr. Janet Zoldan). Lentiviral particles were then harvested, filtered, and incubated with human retinal pigmented epithelial (RPE) recipient cells (ARPE-19, purchased from American Type Culture Collection). Cells were incubated with 2 µg/mL puromycin for one week to select for transduced cells which were then used to generate the monoclonal cell line stably expressing BFP-CLC.