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Vol. 9, Issue 7, 1919-1938, July 1998
Department of Structural Analysis, National Cardiovascular Center
Research Institute, Suita, Osaka 565-8565, Japan
Stress fibers were isolated from cultured human foreskin
fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with
a low-ionic-strength extraction solution and then further extracted
using detergents. With gentle washes by pipetting, the nucleus and the
apical part of cells were removed. The material on the culture dish was
scraped, and the freed material was forced through a hypodermic needle
and fractionated by sucrose gradient centrifugation. Isolated,
free-floating stress fibers stained brightly with fluorescently labeled
phalloidin. When stained with anti-
-actinin or anti-myosin, isolated
stress fibers showed banded staining patterns. By electron microscopy,
they consisted of bundles of microfilaments, and electron-dense areas
were associated with them in a semiperiodic manner. By negative
staining, isolated stress fibers often exhibited gentle twisting of
microfilament bundles. Focal adhesion-associated proteins were also
detected in the isolated stress fiber by both immunocytochemical and
biochemical means. In the presence of Mg-ATP, isolated stress fibers
shortened, on the average, to 23% of the initial length. The maximum
velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral
contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin
light chain kinase inhibitor, KT5926, inhibited isolated stress fiber
contraction. Our study demonstrates that stress fibers can be isolated
with no apparent loss of morphological features and that they are truly
contractile organelle.
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