Our reconstitution system consisted of thin actomyosin layers coupled to SLBs via linker proteins, as described in our earlier work (12). Briefly, actin and myosin were purified from fresh chicken skeletal muscles. HYE and capping protein were expressed in BL21EDE3* bacterial cells and purified by Ni2+ affinity and gel filtration chromatography. SLBs were prepared by fusing small unilamellar vesicles (SUVs) dioleoylphosphatidylcholine (DOPC) [98 mole percent (mol %)] and Ni-NTA-DGS 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (2 mol %) inside small artificial chambers glued on top of clean coverslips (purchased from Avanti Polar). Actin was polymerized by mixing dark and Atto 633 maleimide (AttoTech)–labeled G-actin in a 90:10 ratio, along with capping protein, ATP (Sigma-Aldrich), and 1× KMEH buffer to generate filaments of controlled average length. Atto 565 maleimide–labeled myosin minifilaments were added along with ATP on SLB-tethered actin filaments to study its effect on actin filament organization. The three distinct actomyosin patterns were obtained as a function of myosin density, F-actin density, and F-actin length, as previously reported.

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