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.
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Figure 1 (200 K) - Video-enhanced phase contrast microscopy of living subtype 1 cell showing the radial nature of the actin cytoskeleton in a cell undergoing retrograde flow.
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Figure 2 (57 K) - Immunoblot of anti-sea urchin egg myosin II heavy chain against high-speed supernatant samples from sea urchin eggs and coelomocytes (C-cytes) run on 4% gels.
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Figure 3 (114 K) - Immunolocalization of actin and myosin II in subtype 1 cells
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Figure 4 (86 K) - Immunofluorescent labeling of actin and tubulin in coelomocyte subtypes
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Figure 5 (143 K) - TEM of a critical point-dried and rotary-shadowed cytoskeleton from a subtype 1 coelomocyte
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Figure 6 (171 K) - TEM of critical point-dried and rotary-shadowed cytoskeletons from gelsolin-extracted subtype 1 coelomocytes
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Figure 7 (114 K) - Video-enhanced microscopy of subtype 1 coelomocyte treated with 1 µM cytochalasin D
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Figure 8 (143 K) - Video-enhanced microscopy of a control subtype 1 coelomocyte and the same cell after exposure to 2 µM staurosporine for 15 min
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Figure 9 (143 K) - Retrograde flow rate of control cells and cells treated with kinase inhibitors, as well as retrograde retraction of the central cytoskeleton in cells treated with cytochalasin D
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Figure 10 (95 K) - Video-enhanced microscopy of a control subtype 1 coelomocyte and the same cell treated for 5 min with 15 mM BDM
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Figure 11 (86 K) - Immunolocalization of actin and myosin in coelomocytes fixed in the presence of 15 mM BDM or after washing out the drug for either 5 min or 20 min
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