Force measurements by micromanipulation of a single actin filament by glass needles

Abstract

Single actin filaments (approximately 7 nm in diameter) labelled with fluorescent phalloidin can be clearly seen by video-fluorescence microscopy. This technique has been used to observe motions of single filaments in solution and in several in vitro movement assays. In a further development of the technique, we report here a method to catch and manipulate a single actin filament (F-actin) by glass microneedles under conditions in which external force on the filament can be applied and measured. Using this method, we directly measured the tensile strength of a filament (the force necessary to break the bond between two actin monomers) and the force required for a filament to be moved by myosin or its proteolytic fragment bound to a glass surface in the presence of ATP. The first result shows that the tensile strength of the F-actin-phalloidin complex is comparable with the average force exerted on a single thin filament in muscle fibres during isometric contraction. This force is increased only slightly by tropomyosin. The second measurement shows that the myosin head (subfragment-1) can produce the same ATP-dependent force as intact myosin. The magnitude of this force is comparable with that produced by each head of myosin in muscle during isometric contraction.