Detection of small differences in actomyosin function using actin labeled with different phalloidin conjugates.

Abstract

This study shows that there is only a negligible difference in actomyosin function in the in vitro motility assay among actin filaments labeled with Rhodamine phalloidin (RhPh), Alexa-488 phalloidin (APh), and biotin-XX phalloidin (BPh). Similar results were obtained at varying ionic strengths (0.02-0.13 M), in the presence of imidazole or 3-[N-morpholino]propanesulfonic acid (MOPS) buffer, and at varying MgATP concentrations (0.1-3 mM). If RhPh- and APh-labeled filaments were studied in a given flow cell, there was minimal variability in sliding velocity between the fluorophores (standard deviation of 3% of the absolute sliding velocity). The variability was considerably smaller than that between flow cells, allowing us to use dual labeling of different actin types and then apply analysis of variance to detect minor functional differences between them. Using this method, we could statistically verify a 4% difference (P<0.001) in sliding velocity (3mM Mg ATP) between cardiac and skeletal muscle actin. Suggested improvements of the method would readily allow the detection of even smaller differences. We discuss implications of the results for nanotechnological applications, understanding actomyosin function, and reducing experimental costs and the use of laboratory animals.