Nbd pe

Uptake of NBD labeled phosphatidylserine or phosphatidylethanolamine was examined based on methods described in Viet et al.^{58 (link)}. Briefly, overnight cultures were diluted to an OD₆₀₀:0.2 in RPMI 1640 medium with 165 mM MOPS, pH 7 and grown for 18 hours at 30 °C in ambient or 5% CO₂ shaking conditions. Cells were harvested, washed and resuspended to OD₆₀₀:8 in PBS. 250 μL of cells were aliquoted into a round-bottom 96-well plate for each condition to be tested in triplicate, then incubated at 30 °C for 10 minutes. 1.5 μL of 10 mM NBD-PS (catalog no. 810192P, Avanti Polar Lipids) or 10 mM NBD-PE (catalog no. 810151P, Avanti Polar Lipids) was added to cells and incubated for 30 minutes at 30 °C. After incubation, cells were washed three times with PBS + 4% BSA to extract free NBD-lipids and examined by flow cytometry on an Attune NxT Flow Cytometer with CytKick autosampler and Attune Cytometric software. Gating strategy was optimized for single cells. 100,000 single cells were acquired per sample. Mean fluorescence intensity was determined for each sample.

All reagents were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) unless otherwise stated. DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), CL (cardiolipin), NBD-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), Rhod-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), were from Avanti Polar Lipids (Alabaster, AL, USA). Chemical structures of polymer and lipids used are provided in Figure A1.

DOPG (1,2-dioleoyl-sn-glycero-3-phosphoglycerol), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), MGDG (monogal actosyldiacylglycerol) and DGDG (digalactosyldiacylglycerol), as well as the fluorescence dyes NBD-PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) and LissRhodPE [Lissamin Rhodamin PE; 1,2-Dioleoylsn-glycero-3-phosphoethanolamin-N-(lissamin-rhodamin-B-sulfonyl)] were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL, United States).
The organic solvent (chloroform/methanol 2:1 (v/v)) was removed under a gentle stream of nitrogen gas and overnight vacuum desiccation to allow the formation of a lipid film. Lipids were hydrated in 20 mM HEPES, pH 7.6 buffer and unilamellar liposomes were prepared by five cycles of freezing in liquid nitrogen and thawing at 37°C, followed by 15 extrusions through a 100 nm filter (Nucleopore Track-Etch Membrane, Whatman, Sigma-Aldrich, Taufkirchen, Germany), using an extruder (Avanti Polar Lipids, Inc., Birmingham, AL, United States).

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (SAPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LR-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). NBD-PE (0.5 mol%) was included only in the t-SNARE SBL to test SBL fluidity using fluorescence recovery after photobleaching (Karatekin and Rothman, 2012 (link); Nikolaus and Karatekin, 2016 (link)).

Synthetic 1, 2-dioleoyl-sn-glycero-3-phospho-l-serine (phosphatidylserine, PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (phosphatidylethanolamine, PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine, PC), 1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N-(7-nitro-2–1, 3-benzoxadiazol-4-yl) (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (rhodamine-PE) were obtained from Avanti Polar Lipids. Protein-free SUVs (small unilamellar vesicles) were prepared as follows: lipids (15% PS+30% PE+ 55% PC or 25% PS+30% PE+45% PC, as indicated) were mixed and dried under nitrogen, and lyophilized for 1 h. The dried lipid film was resuspended in Tris buffer (50 mM Tris, pH 7.4, 150 mM KCl) and extruded 20 times through 100 nm polycarbonate filters. SNARE-bearing SUVs were prepared14 (link) using 15% PS+30% PE+55% PC for t-SNARE vesicles (Tr) and 15% PS+27% PE+55% PC+1.5% NBD-PE+1.5% rhodamine-PE for v-SNARE vesicles (Vr).

Lipids dissolved in chloroform were mixed at the desired molar ratio, and the solvent was evaporated by nitrogen gas. The lipid was hydrated with buffer B (20 mM HEPES pH 7.0 and 150 mM NaCl). After five freeze and thaw cycles with liquid nitrogen, liposomes were extruded through a mini-extruder (Avanti Polar Lipids) with 100 nm polycarbonate filter^{56 (link)}.
All lipids used in this study were purchased from Avanti Polar Lipids: PS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), PC (1,2-dioleoylsn-glycero-3-phosphocholine), PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), PA (1,2-dioleoyl-sn-glycero-3-phosphate), PI3P (1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′-phosphate)), PI4P ((1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate)), Cholesterol, NBD-PE, NBD-PS (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine), NBD-PC, NBD-PA (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphate), NBD-Ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine), NBD-Cholesterol (5-cholesten-3ß-ol 6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproate), Rhod-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), and DGS-NTA(Ni) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]).