Fluorescent Labeling and Polymerization of Myosin II

Myosin II was purified from
rabbit skeletal muscle and fluorescently labeled with DyeLight 488
(Invitrogen, Carlsbad, CA, USA) according to Alvarado and Koenderink.^{66 (link)} Labeled
and unlabeled myosin II were stored separately in myosin storage buffer
(300 mM KCl, 25 mM KH₂PO₄, 0.5 mM DTT, 50% (v/v)
glycerol, pH 6.5), where the high ionic strength prevents myosin self-assembly
into bipolar filaments. For experiments, myosin II was dialyzed overnight
in glycerol-free myosin buffer (300 mM KCl, 20 mM imidazol, 4 mM MgCl₂, 1 mM DTT, pH 7.4) and controlled self-assembly into bipolar
filaments was induced by adjusting a KCl concentration of 50 mM via
mixing with myosin polymerization buffer (20 mM imidazol, 1.6 mM MgCl₂, 1 mM DTT, 1.2 mM Trolox, pH 7.4). After an incubation time
of 10 min at 20 °C, the bipolar myosin II filaments were immediately
used for contractile experiments. For the contraction experiments,
the F-actin networks were transferred into an actomyosin buffer by
a 10-fold buffer exchange (50 mM KCl, 20 mM imidazol, 2 mM MgCl₂, 1 mM DTT, 1 mM Trolox, pH 7.4). The reorganization of the
networks was performed at a final ATP concentration of 0.1 mM combined
with an ATP-regeneration system of creatine phosphate (10 mM)/creatine
kinase (0.1 mg/mL)^{66 (link)} and a myosin II concentration
of 0.4 μM.