Csu x1 confocal head

Experimental chambers were assembled by sandwiching two coverslips using parafilm. Before use, the chambers were passivated with a β-casein solution at a concentration of 5 mg/ml in PBS for at least 5 min at RT. GUVs were incubated with 0.2 μM BLOC-1 in the experimental chambers for at least 15 min at RT before observation. To mix GUVs with BLOC-1, we first added 8 μl of the GUVs in 15 μl of an “outer buffer” (60 mM NaCl, 20 mM Tris, pH 7.5), followed by adding 2 μl of BLOC-1 (2.5 μM in stock). Samples were observed using a spinning disk confocal Nikon eclipse Ti-E microscope equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon) and a CMOS camera, Prime 95B (Photometrics). After formation of tubules, sample was pipetted off and flash-frozen for cryo-EM experiments.

For all experiments, GUVs composed of brain total lipid extract (36 ) supplemented with 5 mole percentage (mol%) brain PI(4,5)P₂, 0.2 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], and 0.5 mol% BODIPY-TR ceramide were prepared by electroformation on platinum electrodes overnight at 4°C in a physiologically relevant salt buffer. The salt buffer outside GUVs was 20 mM Tris (pH 7.5), 60 mM NaCl, and 100 mM sucrose. The salt buffer inside GUVs was 20 mM Tris (pH 7.5), 60 mM NaCl, and 100 mM glucose.
GUVs were incubated with IRSp53 I-BAR domain at a bulk concentration of 0.02–0.1 μM for at least 30 min at room temperature before observation. For all experiments, microscope slides and coverslips were washed with water and ethanol followed by passivation with a β-casein solution at a concentration of 5 mg/mL for at least 5 min at room temperature. GUVs were observed by Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon), and QuantEM:512SC camera (Photometrics, Tucson, AZ).