Nbd cholesterol

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]).

Rabbit polyclonal anti MIGA2 antibody (ab 122713) was purchased from Abcam. Rabbit monoclonal anti β-actin antibody was from Cell Signaling Technology (4970; RRID:AB 2223172). Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110). Palmitic acid (P0500) was ordered from Sigma-Aldrich. Sodium dithionite was from Sigma-Aldrich (157953). Etomoxir was purchased from MedChemExpress (HY-50202). The Hela cell line (ATCC #CCL-2; RRID: CVCL 0030) was a gift from Mals Mariappan (Yale University, New Haven, CT), C. elegans cDNA was a gift from Daniel Colon-Ramos (Yale University, New Haven, CT; Xuan et al., 2017 (link)). Plasmid for the 6xhis-PH-tethering construct (Bian et al., 2018 (link)) was a gift from Pietro De Camilli (Yale University, New Haven, CT).

Lipids POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), C1P 18:1/d18:1 (N-oleoyl-ceramide-1-phosphate) and C1P 16:0/d18:1 (N-palmitoyl-ceramide-1-phosphate) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Fluorophores NBD-Cholesterol and TopFluor-C1P were purchased from Avanti Polar Lipids (Alabaster, AL, USA), fast DiO was purchased from ThermoFisher Scientific (Massachusetts, USA) and both Atto550-DMPE and Atto488 DOPE were purchased from Sigma Aldrich (Missouri, USA). Structures of lipids and ceramides are presented in Fig. S11. Doodle of fluorophores showing binding position is presented in Fig. S12. Sucrose and glucose were purchased from Sigma Aldrich (Missouri, USA). Low-melting temperature agarose polymer ( $T_m$  65  ${}^{\circ }$ C, $T_g$  25  ${}^{\circ }$ C) was purchased from Fisher Scientific (Massachusetts, USA). The ultra-pure water used in experiments was obtained from a water purification system (Millipore).