Force Generation by Membrane-Bound Myo1c, a Single Molecule Study

Abstract

Myosin-IC (Myo1c) is a class-1 myosin that links cell membranes to the actin cytoskeleton. Actin binding occurs via the motor domain, while the tail domain interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) through a pleckstrin homology domain. We expressed recombinant full-length myo1c and we showed previously that it is able to propel actin filaments while attached to a fluid supported-lipid-bilayer containing PtdIns(4,5)P2 in an in vitro gliding assay. However, Myo1c in this ensemble assay undergoes its working stroke under very low-loads, and it is of interest to measure the ability of the motor to generate force while bound to a fluid bilayer. Therefore, we measured the working stroke of membrane-bound myo1c molecules using an optical tweezers in the three-bead assay configuration. We engineered spherical supported bilayers as pedestal beads with membranes consisting of 98% DOPC and 2% PtdIns(4,5)P2. Membrane fluidity of the bilayers was confirmed by FRAP of labeled lipids. We detected actin attachments of single membrane-bound Myo1c molecules. We observed unitary work stroke events against the force of the trap, followed by the relaxation of tension presumably due to diffusion of the Myo1c on the lipid bilayer. The size of the working stroke of membrane-bound Myo1c was reduced compared to Myo1c on a solid substrate, possibly due to the elasticity of the lipid membrane. The subsequent relaxation of the tension takes place within 100 ms of the working stroke. Our experimental results show that Myo1c can develop tension on fluid membranes and may therefore actively contribute to tension between the membrane and actin, as well as to powering changes in membrane morphology. This assay will be useful to investigate the mechanical properties of the large number of cytoskeletal proteins that are proposed to link membranes and actin. Supported by NIH RO1GM57247.