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* The Randall Institute, Kings College London, University of London, London WC2B 5RL, United Kingdom; and We have determined the structural organization and dynamic behavior of actin filaments in entire
primary locomoting heart fibroblasts by S1 decoration,
serial section EM, and photoactivation of fluorescence.
As expected, actin filaments in the lamellipodium of
these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are
two observable types of actin filament organization. A
less abundant type is located on the inner surface of the
plasma membrane and is composed of short, overlapping actin bundles (0.25-2.5 µm) that repeatedly alternate in polarity from uniform barbed ends forward to
uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells
(PtK2 cells) and to the known organization of muscle
sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly
ventrally located and is composed of long, overlapping
bundles (average 13 µm, but can reach up to about 30 µm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In
each actin bundle, polarity gradually changes along the
length of the bundle. Actual actin filament polarity at
any given point in the bundle is determined by position
in the cell; the closer to the front of the cell the more
barbed ends of actin filaments face forward.
By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the
lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin
filaments: one remains stationary and the other moves
forward with respect to substrate at the rate of the cell
body.
This is the first time that the structural organization
and dynamics of actin filaments have been determined
in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.
Department
of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0450
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