Imaging of thermal activation of actomyosin motors

Abstract

We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with rise and fall times of several ms, and locally in a region of approximately 10 m in diameter with a temperature gradient up to 2°C͞m. Temperature distribution was imaged pixel by pixel by image processing of the f luorescence intensity of rhodamine phalloidin attached to (single) actin filaments. With short pulses, actomyosin motors could be activated above physiological temperatures (higher than 60°C at the peak) before thermally induced protein damage began to occur. When a sliding actin filament was heated to 40-45°C, the sliding velocity reached 30 m͞s at 25 mM KCl and 50 m͞s at 50 mM KCl, the highest velocities reported for skeletal myosin in usual in vitro assay systems. Both the sliding velocity and force increased by an order of magnitude when heated from 18°C to 40-45°C. Temperature-pulse microscopy is expected to be useful for studies of biomolecules and cells requiring temporal and͞or spatial thermal modulation.Recent advances in optical microscopic techniques have made it possible to image single protein molecules in solution (1, 2) and investigate the dynamic nature of molecular motors (3-8).To introduce an additional dimension to this technology, we have developed temperature-pulse microscopy (TPM), in which a microscopic sample(s) in aqueous solution is heated reversibly.There have been several reports on the effects of temperature jumps under an optical microscope: for example, on physiological functions of muscle fibers (9-12) and on phase transition phenomena in membranes of phospholipid vesicles and cells (13). To prevent thermal deterioration of biological samples, and to confirm the absence of the deterioration, it is highly desirable to restore the starting temperature as soon as the measurement is finished. In our TPM, temperature is elevated spatially and temporally by illuminating a lump of metal particles by IR laser; a concentric temperature gradient is created around the lump of metal particles. When the laser beam is shut off, the heat is rapidly dissipated into the surrounding medium. Thus, a square-wave temperature pulse with rise and fall times of less than 10 ms is generated. Exposure to high temperature is minimized, and repetitive thermal cycling is easily programmed. The local heating also permits simultaneous observation of the sample behaviors at various temperatures.In the microscopic temperature-imaging techniques reported so far, the temperature was estimated either from the thermal quenching of fluorescence (14-16) or from the thermal shift of the fluorescence spectrum (13). Here, we applied the former technique. In our TPM, a concentric temperature gradient is formed around the metal aggregate, as assessed from thermal quenching of a fluorescent dye bound to actin filaments with a slope of 1-2°C͞m and extension out to 10-20 m. The temperature distribution on single actin filaments also could be imaged.We have applied ...

Highly thermosensitive Ca2+dynamics in a HeLa cell through IP3receptors

“…Production of heat pulse and measurement of quick temperature changes in the cytoplasm Microscopic heat pulse (rectangular temperature change) was produced by an infrared laser beam focused on an agglomeration of metal nanoparticles at the tip of a glass micropipette placed near single living HeLa cells, such that the local temperature gradient within several tens of micrometers was created in solution (Kato et al, 1999;Zeeb et al, 2004) [ Fig. 1(A)].…”

Bead size effects on protein‐mediated DNA looping in tethered‐particle motion experiments

“…Typical examples are protein binding and dissociation rates or velocities of motor molecules. [3][4][5] While such experiments can be performed with small markers such as gold nanoparticles, in practice, a larger marker microsphere with a radius of several hundreds of nanometers is often preferred because good image quality can be readily achieved with an inexpensive and robust bright-field microscopy set-up. Imaging nanoparticles, on the other hand, requires cumbersome dark-field imaging and an expensive low-light camera.…”

Carbon Nanotube–Polymer Composite for Light‐Driven Microthermal Control

“…We prepared an SWNT hybrid microchip to investigate the photoinduced exothermic properties of the PL-BSA-SWNT-PDMS composite (see Figure 3 a, b and the Supporting Information for details). The temperature distribution in a microchannel was measured by determining the degree of fluorescence intensity of 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA), [22] which is a temperature-responsive reagent in a reversible fashion, upon NIR irradiation (Figure 3 c). An aqueous solution of .…”