FLEXIBILITY OF ACTIN-FILAMENTS DERIVED FROM THERMAL FLUCTUATIONS - EFFECT OF BOUND NUCLEOTIDE, PHALLOIDIN, AND MUSCLE REGULATORY PROTEINS

Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy , The persistence length (L(p)) was derived from the analysis of eithe r the cosine correlation function or the average transverse fluctuatio ns of a series of recorded shapes of filaments assembled from rhodamin e actin. Phalloidin-stabilized filaments had a persistence length of 1 8 +/- 1 mu m, in agreement with recent observations, In the absence of phalloidin, rhodamine-labeled filaments could be observed under a var iety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same L(p) of 9 +/- 0.5 mu m, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of t he tightly bound divalent metal ion, In the presence of BeF3-, which m imics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably mor e rigid, with L(p) = 13.5 mu m. Hence, newly formed F-ADP-P-i-actin fi laments are more rigid than ''old'' F-ADP-actin filaments, a fact whic h has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (L(p) = 20 +/- 1 mu m) in the off state (-Ca2+), and flexible (L(p) = 12 mu m) in the on state (+Ca2+), consistent with the steric blocking model, In a greement with x-ray diffraction data, no appreciable difference was re corded between the off and on states using smooth muscle tropomyosin a nd caldesmon (L(p) 20 +/- 1 mu m). In conclusion, this method allows a ccurate measurement of small (less than or equal to 15%) changes in me chanical properties of actin filaments in correlation with their biolo gical functions.