Looped Actin Structures for Testing Processive Molecular Motors

A method is described for constructing looped actin filaments. Circular islands of nitrocellulose were patterned onto a microscope cover slip by micro-contact printing. A flow cell was then constructed over the patterned surface. Skeletal myosin was introduced into the assay which non-specifically attached to the nitrocellulose islands. Labeled actin filaments were added to the assay and self assembled along the perimeter of the islands to form circular loops of around 4-6 mum in diameter. By altering the biochemical conditions in the assay, the bound myosin acted as a linker to hold the assembled filament in this looped configuration. Regions of the loop with enhanced fluorescent intensity suggest "closed loops" were formed from single filaments with overlapping ends. Adaptations of this assembly technique can be used for in vitro motility assays with highly processive actin based motor proteins such as myosin V. A more complete understanding of the factors that influence the processivity of myosin V may ultimately assist in efforts to exploit the molecule's unique functionality in engineered micro- and nano-fluidic devices