Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay

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. Electronic supplementary material The online version of this article (10.1007/s10974-019-09505-1) contains supplementary material, which is available to authorized users.

Multistep orthophosphate release tunes actomyosin energy transduction

“…Second, the independence between experimental occasions and myosin batch is supported by our data in Supplementary Figs. 16, 18 as well as previous data showing very similar kinetic properties of myosin from different preparations 76 , 102 . Accordingly, we found similar results in three hs-AFM experiments performed on different days using different surface preparations, solutions and HMM from two different myosin preparations.…”

“…In the replication of single-molecule fluorescence experiments, each binding event of a fluorescent ATP molecule to a myosin molecule was assumed to be an independent random event independent of the experimental occasion, or myosin batch as supported by previous findings of very similar kinetic properties of myosin from different preparations 76 , 102 . Furthermore, the assumption is supported by similar results in experiments performed on different days and/or using different myosin preparations (Fig.…”

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“…No sample size calculation was performed prior to the experiments. Instead we relied on previous information 7 , 80 .…”

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“…Finally, assay solution was prepared by adding 0.2 mg mL À1 creatine phosphokinase (CPK), 2.5 mM creatine phosphate (CP), 1 mM magnesium adenosine triphosphate (MgATP), an oxygen scavenger mixture (GOC): 3 mg mL À1 glucose : 0.1 mg mL À1 glucose oxidase, 0.02 mg mL À1 catalase, 10 mM DTT, and 45 mM (KCl) to the LISS solution, giving a final ionic strength of 60 mM. [45,46] In Vitro Motility Assays: HMM, fluorescently labeled actin and nonfluorescent-blocking actin, [47] were diluted in the wash solution. To perform the in vitro motility assay, flow cells were built using a glass coverslip and a nanostructured chip on top, separated with Parafilm spacers.…”