Michael Anson1 and Monica Canepari2
1National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom, 2 Instituto di Fisiologia Umana, Universitaí di Pavia, Via Forlanini 6, 27100 Pavia, Italy.
We have examined the effects of temperature on the motility and velocity of Ca2+-regulated actin filaments. F-actin was incubated on ice with a molar excess of purified tropomyosin and troponins and labelled with rhodamine-phalloidin. Rabbit skeletal HMM was bound to nitrocellulose-coated glass coverslips which formed the base of the flow-cells. After washing with BSA, 10 nM regulated actin filaments were infused and bound in rigor. They were visualized in a fluorescence microscope under laser excitation at 543 nm by an intensified CCD camera and the images stored on S-VHS video tape for off-line computer analysis. At 150 mM ionic strength, 0.35 - 0.7% methyl cellulose and 1 mM EGTA at pH 7.2, regulated filaments remained rigor-like in the presence of 2 mM Mg-ATP. Upon increasing the concentration of free Ca2+ to 300 µM (pCa 3.5 ) at 25°C, circa 90% of filaments moved at approximately 3 µm s-1. As the temperature was decreased, the filament velocity reduced rapidly until below 17 °C sliding movement was not apparent. Analysis showed that the average velocity was less than 40 nm s-1 at 17 °C. On increasing the temperature the filaments commenced moving at 20 °C and were again translating with near-normal velocity at 25 °C. Substituting actin filaments without regulatory proteins, a less dramatic temperature dependence was observed; filament velocity at 25 °C was 3 µm s-1 and at 17 °C decreased to 0.5 µm s-1. These results show stronger temperature dependencies of velocity in vitro than previously reported for unregulated actin at low ionic strength.