A.M. Gordon, M.A. LaMadrid, Y. Chen, Z. Luo and P.B. Chase
Department of Physiology and Biophysics and Department of Radiology, University of Washington, Seattle, WA 98195.
Using an in vitro motility assay, we investigated Ca2+ regulation of sliding of thin filaments reconstituted from rabbit fast skeletal actin (labeled with rhodamine-phalloidin), troponin (Tn), and tropomyosin (Tm) on nitrocellulose surfaces coated with rabbit fast skeletal HMM. Filaments were visualized by epifluorescence and sliding measured for different [Ca 2+]'s at 30°C, 2 mM MgATP, ionic strengths (g) of 0.085, 0.115 and 0.14 M, 0.4-0.7% methylcellulose, and added Tm-Tn. Intensified images were recorded on tape and analyzed using a Motion Analysis System. In contrast to F-actin (for which speed was unaffected by [Ca2+]), the speed increased in a graded manner with [Ca2+] while the number of regulated thin filaments moving was more steeply regulated with none moving without Ca2+ to nearly 100% moving with Ca2+. Increased g shifted both relationships towards higher [Ca2+]. Analysis of data (0.14 M, 250 mg/ml HMM, pCa 4-9.2) for length dependence of speed and fraction moving shows no significant differences among filaments longer than approximately 3 um. Filaments shorter than approximately 3 um displayed slower average speeds and moving fractions; however, subtraction of non-moving filaments or removal of filaments with SD/Ave 0.5 showed that these shorter filaments moved at similar speeds as the longer filaments, implying that filaments shorter than 3 um have a higher probability of not moving or moving in a saltatory manner. The force-pCa relationship for rabbit psoas skinned fibers obtained under similar conditions (30 °C, 0.14M g) paralleled the speed-pCa relationship for the in vitro regulated filaments. These results suggest that relatively few crossbridges are needed to make filaments move, but that many have to be cycling to make the regulated filament move at maximum speed. (Supported by NIH HL52558.)