Abstract
The in vitro motility assay allows studies of muscle contraction through observation of actin filament propulsion by surface-adsorbed myosin motors or motor fragments isolated from muscle. A possible problem is that motility may be compromised by nonfunctional, “dead”, motors, obtained in the isolation process. Here we investigate the effects on motile function of two approaches designed to eliminate the effects of these dead motors. We first tested the removal of heavy meromyosin (HMM) molecules with ATP-insensitive “dead” heads by pelleting them with actin filaments, using ultracentrifugation in the presence of 1 mM MgATP (“affinity purification”). Alternatively we incubated motility assay flow cells, after HMM surface adsorption, with non-fluorescent “blocking actin” (1 µM) to block the dead heads. Both affinity purification and use of blocking actin increased the fraction of motile filaments compared to control conditions. However, affinity purification significantly reduced the actin sliding speed in five out of seven experiments on silanized surfaces and in one out of four experiments on nitrocellulose surfaces. Similar effects on velocity were not observed with the use of blocking actin. However, a reduced speed was also seen (without affinity purification) if HMM or myosin subfragment 1 was mixed with 1 mM MgATP before and during surface adsorption. We conclude that affinity purification can produce unexpected effects that may complicate the interpretation of in vitro motility assays and other experiments with surface adsorbed HMM, e.g. single molecule mechanics experiments. The presence of MgATP during incubation with myosin motor fragments is critical for the complicating effects.
Highlights
Cyclic interactions between the molecular motor myosin II and actin filaments underlie cell movement such as muscle contraction
To the best of our knowledge, it has not been studied previously how the presence of MgATP during heavy meromyosin (HMM) incubation might affect function. We investigated this issue while comparing the affinity purification procedure to the use of blocking actin with regard to improvement in motile function in the in vitro motility assays with HMM adsorbed to TMCSderivatized or nitrocellulose coated glass surfaces
Whereas both procedures increased the fraction of motile filaments, our results show that the affinity purification procedure has the undesirable potential to reduce sliding velocity
Summary
Cyclic interactions between the molecular motor myosin II and actin filaments underlie cell movement such as muscle contraction. The “blocking actin” filaments act as barriers against the interaction between dead heads and fluorescent actin filaments Both ‘affinity purification’ and an incubation step with ‘blocking actin’ are procedures commonly used for improving the observed actin-myosin function in the in vitro motility assay. In order to achieve this type of characterization, we here performed in vitro motility assays to compare and analyze the effects of ‘affinity purification’ and ‘blocking actin’ on actin-myosin motility in the IVMA Whereas both procedures increased the fraction of motile filaments to different degrees on different surface substrates we were surprised to find that the actin sliding speed often was reduced by the affinity purification procedure but not by the use of blocking actin. This warrants further detailed investigations in the future because different experimental findings may result from different surface adsorption mechanisms of the motors, possibly influencing estimates of myosin step length, stiffness, average force etc
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