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Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.

We have combined the simplicity of the periodic pattern method of photobleaching with the signal processing advantage of scanning detection. A periodic photobleaching pattern is produced in the sample by brief, intense illumination through a grating. The grating is then translated at constant speed through the reference beam to produce a moving pattern on the specimen. A modulation of fluorescence emission is produced as the bleached pattern and the illumination pattern fall into and out of phase. The resulting photocurrent contains an ac component whose frequency is determined by the spacing and velocity of the grating and whose amplitude relative to the dc component is determined by the extent of photobleaching. The decay constant of the ac component of the photocurrent is determined by the spacing of the grating and the diffusion coefficient of the labeled species. The major distinct advantages of the technique are: (1) the decay at the fundamental frequency is observed in real time, (2) adventitious photobleaching during the monitoring phase of the measurement does not perturb the determined parameters, (3) the measurement is insensitive to dc drift components, and (4) the measurement is insensitive to motion of the specimen. These substantial advantages are implemented by straightforward modifications in standard methodology.

We have combined fluorescent analogue cyto-..chemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine -serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3.of that observed in aqueous solution;'90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and r10% of the actin has a mobility comparable to that of F-actin in aqueous solution.Therefore, no more than' 10% of .the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection ofphalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different'parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm; we found the low-.mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.

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