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

Chemical reagents MOPS (3-(N-morpholino)propanesulphonic acid), HEPES (4-(2-hydroxyethyl)-1piperazineethanesulphonic acid), Tris hydrochloride, NaCl, DTT (dithiothreitol), IPTG (isopropyl-β-D-thiogalactopyranoside), β-mercaptoethanol, thrombin protease, and Triton X-100 were purchased from Fisher Scientific. EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), TCEP (tris(2-carboxyethyl)phosphine hydrochloride), PMSF (phenylmethanesulfonyl fluoride), EDTA-free protease inhibitor tablets, Thrombin CleanCleave Kit, PLL (poly-L-lysine), Atto 488 NHS-ester, and Atto 594 NHS-ester were purchased from Sigma-Aldrich. HRV-3C (human rhinovirus-3C) protease, neutravidin, Oregon Green 488-DHPE (Oregon Green 488 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine), and Texas Red-DHPE were purchased from Thermo Fisher Scientific. mPEG-SVA (mPEG-succinimidyl valerate), biotin-PEG-SVA, mPEG-silane, and biotin-PEG-silane (all PEGs were molecular weight 5,000 Da) were purchased from Laysan Bio (Arab, AL, USA). DP-EG10-biotin (dipalmitoyl-decaethylene glycol-biotin) was generously provided by Dr. Darryl Sasaki of Sandia National Laboratories, Livermore, CA (Momin et al., 2015) . All other lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA), including: PtdIns(4,5)P 2 (L-αphosphatidylinositol-4,5-bisphosphate, from porcine brain), DOGS-NTA-Ni (1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], nickel salt), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt). The lipid compositions for all experiments are listed in the figure captions.