Liposome preparation and myoVa conjugation

Phospholipid liposomes of 350 nm diameter, composed of (molor ratio) 84 parts DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 5 parts PEG-ylated phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000), 5 parts cholesterol, 5 parts 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MBP:PE) and 1 part carbocyanine dye DiI or DiO Cell-labelling Solution (Thermo Fisher Scientific) were created through extrusion using filter membranes¹⁷. In brief, the lipid mixture was mixed then dried under a nitrogen stream followed by 1 h under vacuum (Rotovap; Eppendorf). The mixture was then rehydrated to 5 mg ml⁻¹ using PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM N KH2PO4) at pH 7.2. Using an Avanti Mini-Extruder (Avanti Polar Lipids), the liposomes were then extruded for 20 passes using a 1 μm pore diameter filter (Whatman). Liposomes were mixed with a final concentration of 1 μM thiolated Neutravidin (SH-NaV) and incubated at room temperature for 1 h to covalently conjugate the maleimide moiety of the MBP:PE within the liposome membrane to the thiol groups of the SH-NAV¹⁷. After incubation, excess SH-NAV was removed by centrifugation at 420,000 g for 10 min. Liposomes were re-suspended in PBS (pH 7.2) and extruded for 20 passes through a membrane with pore size specific to the desired final liposome diameter, which varied between 100 and 650 nm (Supplementary Fig. 1). A double headed, heavy meromyosin myosin Va (myoVa) construct was tagged at the C-terminus with an 88-aa biotin ligase recognition sequence²⁵. The myoVa was coexpressed with calcium-insensitive calmodulin light chain using a baculovirus/Sf9 cell system and was purified using affinity chromatography to a C-terminal FLAG tag on the myosin heavy chain^26,27. MyoVa motors were bound to the liposome exterior surface through the C-terminal biotin on the myoVa construct binding to the SH-NaV, which is bound to the liposome¹⁷. Specifically, motors (3.3 μl, 500 nM) were conjugated to 350 nm DOPC liposomes (10 μl of 3.9 nM) with 6.7 μl of buffer (25 mM imidiazole, 4 mM MgCl₂, 1 mM EGTA, 25 mM KCl, 10 mM DTT, with 1 mg ml-1 bovine serum albumin (BSA)) and incubated for 15 min at room temperature.