One of the most obvious systems to explore at the single-molecule level was the movement of motor proteins, such as kinesin and myosin, along the periodic actin or microtubule filament. The ability to visualize single events would provide crucial insights into the process of chemomechanical transduction within the cell.
Previous biochemical work had suggested that motor proteins bind to a filament, undergo a conformational change driven by nucleotide hydrolysis that displaces the filament by a certain distance and then dissociate. However, the details of the force exerted by the motor, the step size of the motion and the kinetics of each state remained inaccessible. In 1989, Howard, Hudspeth and Vale reported that a single molecule of kinesin, bound to a microscope slide, was capable of propelling a microtubule across the surface. One kinesin molecule moved a microtubule at the same speed as several kinesin molecules. Importantly, the data showed that kinesin was processive, indicating that the two-headed motor might operate by a hand-over-hand mechanism and take hundreds of steps before dissociating.
In 1993, Block and co-workers used optical tweezers to tackle a pressing question in the motor field: does kinesin move along a microtubule with a specific step size? Kinesin was attached to a micron-sized bead, which was held in the focus of a laser trap. The bead was placed in contact with a microtubule fixed on a coverslip, and the movement of kinesin along the microtubule was detected using a sensitive photodetector. By this approach they determined that kinesin moves in quantized steps of 8 nm. Similarly, a dual-beam trap with a suspended actin filament interacting with a single myosin molecule attached to a surface was developed by Spudich and co-workers to measure nanometer displacements and piconewton forces by myosin. They showed that muscle myosin has a step size of 
10 nm, resolving the decade-long controversy surrounding this issue.
Single-molecule assays have revolutionized cytoskeletal research.
Jonathon Howard
Along with step size, energy consumption by a motor during movement was a pressing issue in the field. The technological breakthroughs needed to resolve this were initiated by the Yanagida laboratory. In 1995, this group constructed a microscopic set-up that measured both epifluorescence and total internal-reflection fluorescence (TIRF). Fluorescent dyes bound to myosin subfragments allowed individual molecules to be identified. At the same time, the association of fluorescently-labelled ATP molecules with a single myosin fragment was detected. The dissociation rate determined was identical to the solution measurements, establishing this as a valid approach to characterize ATPases. A year later, the Yanagida laboratory further showed that TIRF could be used to follow the movement of individual fluorescently-labelled kinesin molecules along a filament. Notably, single-headed kinesin was found to be unable to move processively.
These examples highlight the role that single-molecule technologies have had in revealing the microscopic details of motor energetics, forces and movement. They have served as a basis for examining many protein–nucleic-acid interactions as well.
