Abstract
Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.
Highlights
Single actin filaments undergoing brownian movement in two dimensions were observed at 20°C in fluo· rescence optical video microscopy
The values found for the persistence length are in agreement with recently published data for phalloidin-decorated F-actin; we show that in the absence of phalloidin actin filaments are 2-fold more flexible CLp = 9-10 J-Lm)
The persistence length of actin filaments was 18 ± 1 p.m once stabilized by phalloidin
Summary
Single actin filaments undergoing brownian movement in two dimensions were observed at 20°C in fluo· rescence optical video microscopy. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration Such nonstabilized F-ADP-actin filaments had the same Lp of 9 ± 0.5 p.m, whether they had been assembled from ATP-G-actin or from ADP-Gactin, and independently of the tightly bound divalent metal ion. Thymosin i34 allows the formation of a pool of unpolymerized actin [6]; the size of this pool is controlled by the concentration of free G-actin at steady state, which is itself regulated by capping proteins and profilin [7, 8] It is via the concerted control of assembly dynamics and of mechanical properties that the biological functions of actin filaments are fulfilled. Proteins binding to filaments and regulating contractility, such as tropomyosin, troponins, and
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