AJP - Cell Physiology, Vol 262, Issue 3 C569-C577, Copyright © 1992 by American Physiological Society
Distribution of actin filament lengths measured by fluorescence microscopy
S. Burlacu, P. A. Janmey and J. Borejdo
Baylor Research Institute, Baylor University Medical Center, Dallas, Texas 75226.
We analyzed the distribution of actin filament lengths by optical
microscopy (OM). OM avoids possible alterations in the size or structure of
actin filaments occurring during sample preparation for electron microscopy
(EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate
(TRITC)-phalloidin were analyzed for both size distribution and
flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4,
25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM
beta-mercaptoethanol] filaments did not aggregate into bundles and remained
stable at nanomolar concentrations for at least 1 h. At the same
concentration, actin labeled directly with rhodamine (no phalloidin) formed
unstable filaments whose average length decreased with time. The number
average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns,
the ratio (rho) of the weight average length to the number average length
was 2.06, and the correlation length (1/lambda) was 8.33 microns. These
parameters were in good agreement with the values determined by EM for
filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150
column before polymerization did not have a significant effect on the
distribution of lengths but made filaments more stiff (1/lambda = 12.5
microns). Millimolar concentration of ATP increased the correlation length,
and gelsolin had the expected fragmenting effect on filaments. These
results show that OM can be used as a fast and reliable method to analyze
the distribution and flexibility of actin filaments and suggest that, in
spite of extensive manipulation of actin filaments during sample
preparation, EM is a valid tool for determination of size parameters of
actin filaments.
