Unidirectional Movement Of Fascin-induced Actin Bundle On Heavy Meromyosin In An In Vitro Motility Assay

Abstract

PURPOSE: Bionano-motors offer unique promise for the development of nanotechnological devices. Controlling motor protein movement is the key to integrating active transport properties into devices. Here we investigate how actin filament bundling alters filament translational across a micropatterned surface. The myosin-driven movement of actin filaments is random or not unidirectional. We examined whether the ratio of actin to GST-tagged fascin altered the direction of bundles on heavy meromyosin (HMM). METHODS: For motility assay, HMM was adsorbed to nitrocellulose-coated coverslip and incubated with fluorescent labeled actin filament bundles and ATP. Movement of bundles on HMM was observed under a fluorescence microscope. Analysis of bundle translational efficiency (E) was determined from the formula E=100[1-(Da-Ds)/Da], where Ds and Da represent the shortest and the actually moving distances from the start to the end respectively. RESULTS: Decreases in the ratio of actin to fascin were associated with increased translational efficiency (E was 53±6% in R=5:1(g/g), 71±4% in 3:1 and 80±3% in 1.5:1) and increased unidirectionality of bundle movement. Similar results were found irrespective of HMM concentration (90, 120, 150, and 180 ìg/ml HMM). CONCLUSIONS: Altering fascin crosslinker ratio is capable of "tuning" of actin bundle stiffness and HMM-dependent bundle translation. This strategy may be important in the design of nanotechnology devices where myosin is used as a bionanomotor.