Imaging of thermal activation of actomyosin motors

We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with ris e and fall times of several ms, and locally in a region of approximately 10 mu m in diameter with a temperature gradient up to 2 degrees C/mu m, Tempe rature distribution was imaged pixel by pixel by image processing of the fl uorescence intensity of rhodamine phalloidin attached to (single) actin fil aments, With short pulses, actomyosin motors could be activated above physi ological temperatures (higher than 60 degrees C at the peak) before thermal ly induced protein damage began to occur. When a sliding actin filament was heated to 40-45 degrees C, the sliding velocity reached 30 mu m/s at 25 mM KCl and 50 mu m/s at 50 mM KCl, the highest velocities reported for skelet al myosin in usual in vitro assay systems. Both the sliding velocity and fo rce increased by an order of magnitude when heated from 18 degrees C to 40- 45 degrees C. Temperature-pulse microscopy is expected to be useful for stu dies of biomolecules and cells requiring temporal and/or spatial thermal mo dulation.