Biotin pe

POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Cat# 850457), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt, Cat# 840457), biotin-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt), and Cat# 870273) in chloroform were purchased from Avanti Polar Lipids. Streptavidin (Cat# 434301), the membrane dye DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt, Cat# D7757), Vybrant™ DiD Cell-Labeling Solution (Cat# V22887) and RPMI 1640 Medium were purchased from Thermo Fisher Scientific. Chemicals and enzymes including luminol (Cat# 123072), L-012 (8-amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d]pyridazine-1,4-dione, sodium salt) (Cat# SML2236) and phorbol-12-myristate-13-acetate (Cat# P8139) were purchased from Sigma-Aldrich. Ni²⁺-NTA functionalized polystyrene beads (2 μm) were purchased from Micromod Partikeltechnologie GmbH. PE labelled IgG1 mouse anti-human CD11b (eBioscience, Cat# 12-0018) was used. Confocal microscopy experiments with GUVs were performed in 18-well μ-slide glass bottom chambers (Cat# 80827) from ibidi. Bead aggregation experiments were performed using an 8 well μ-slide (Cat# 80826) from ibidi. Buffers and aqueous solutions were prepared with Milli-Q grade water.

PM-mimic acceptor vesicles with reconstituted syntaxin-1A and SNAP-25A were generated as described previously (Diao et al., 2013 (link); Kyoung et al., 2013 (link); Lai et al., 2017 (link), Leitz et al., 2024a , Leitz et al., 2024b ). Briefly, a lipid film of Porcine Total Brain Extract (Avanti Polar Lipids, 131101C), 3% PIP2 (Avanti Polar Lipids, 840046X), 1% Biotin PE (Avanti Polar Lipids, 870285P) and 1% DAG (Avanti Polar Lipids, 800811C) was generated by combining the lipids followed by evaporation under argon. Lipid films were allowed to dry overnight under a vacuum. PM vesicles were reconstituted with syntaxin-1A and SNAP-25A in a VB containing 50 mM of sulforhodamine B (Thermo Fisher Scientific). The protein-lipid mixture was then added to a 5-mL CL-4B (Millipore Sigma GE17-01501-01) column. 200 μL fractions were collected and analyzed on an SDS PAGE gel with Coomassie stain to assess which fractions had visible bands for both syntaxin-1A and SNAP25. Select fractions were pooled and dialyzed overnight at 4 °C against 2 L of VB with Bio-Beads SM-2 resin (BioRad, 1523920). Vesicle size and homogeneity were determined via dynamic light scattering (DLS) using a DynaPro NanoStar (Wyatt Technologies) (Supplementary Fig. 3).

Vesicle solutions containing (i) 0.05% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (biotin-PE, Avanti® Polar Lipids, Inc) mixed with 99.95% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti® Polar Lipids, Inc) or (ii) 5% or 10% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA, Avanti® Polar Lipids, Inc) mixed with 95% or 90% POPC, respectively, were prepared at a concentration of 0.5 mg/mL by the following protocol. The different lipids were first mixed in 100 µL chloroform. To remove the chloroform, the lipids were dried using a N₂ gas flow for 10 minutes. After evaporation of chloroform the vesicles were suspended and thoroughly mixed in 1 mL of filtered (0.2 µm Minisart® Syringe filter, Sartorius) washing buffer: 150 mM NaCl, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, Sigma), pH 7.4 for all lipids. The vesicles were then incubated on ice for 1–2 h followed by tip sonification with a CV18 model tip sonicator (Chemical instruments AB) for 15 minutes with a pulse time of 10 s and an amplitude of 55%. The lipid stock solutions were stored at −20 °C in chloroform and the vesicle solutions at 4 °C until used.