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Vol. 10, Issue 12, 4075-4090, December 1999
and
*Department of Biology, Dickinson College, Carlisle, Pennsylvania
17013; Sea urchin coelomocytes represent an excellent experimental model
system for studying retrograde flow. Their extreme flatness allows for
excellent microscopic visualization. Their discoid shape provides a
radially symmetric geometry, which simplifies analysis of the flow
pattern. Finally, the nonmotile nature of the cells allows for the
retrograde flow to be analyzed in the absence of cell translocation. In
this study we have begun an analysis of the retrograde flow mechanism
by characterizing its kinetic and structural properties. The
supramolecular organization of actin and myosin II was investigated
using light and electron microscopic methods. Light microscopic
immunolocalization was performed with anti-actin and anti-sea urchin
egg myosin II antibodies, whereas transmission electron microscopy was
performed on platinum replicas of critical point-dried and
rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical
actin network, which feeds into an extensive array of radial bundles in
the interior. These actin bundles terminate in a perinuclear region,
which contains a ring of myosin II bipolar minifilaments. Retrograde
flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was
blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin
cytoskeleton to separate from the cell margin and undergo a finite
retrograde retraction. In contrast, inhibition of myosin II function
either with the wide-spectrum protein kinase inhibitor staurosporine or
the myosin light chain kinase-specific inhibitor KT5926 stopped flow
in the cell center, whereas normal retrograde flow continued at the
cell periphery. These differential results suggest that the mechanism
of retrograde flow has two, spatially segregated components. We propose
a "push-pull" mechanism in which actin polymerization drives flow
at the cell periphery, whereas myosin II provides the tension on the
actin cytoskeleton necessary for flow in the cell interior.
Mount Desert Island Biological Laboratory,
Salisbury Cove, Maine 04672; and §Laboratory of Molecular
Biology, University of Wisconsin, Madison, Wisconsin 53706
Corresponding author. E-mail address:
henson{at}dickinson.edu.
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