Background: Directional cell motility implies a steering mechanism and a functional asymmetry between the cell's front and rear. However, how this functional asymmetry arises and is maintained during cell locomotion is unclear. Lamellar fragments of fish epidermal keratocytes present a simplified, perhaps minimal, system for analyzing this problem because they consist of little else than the motile machinery enclosed by a membrane and, yet, can locomote with remarkable speed and persistence.
Results: We have produced two types of cellular fragments: discoid stationary fragments and polarized locomoting ones. Organization and dynamics of the actin-myosin II system were isotropic in stationary fragments and anisotropic in locomoting ones. To investigate if creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin-myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to locomote, and the locomotion and associated anisotropic organization of actin-myosin II system were sustained after withdrawal of the stimulus.
Conclusions: We propose a model in which lamellar cytoplasm is considered a dynamically bistable system,--capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation and translocation reinforces the asymmetry of the machinery favoring further translocation.
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Figure 1 (131 K) - Morphology and motility of keratocyte cytoplasmic fragments
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Figure 2 (147 K) - Cytoskeletal organization of locomoting and stationary keratocyte fragments
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Figure 3 (77 K) - Dynamics of microinjected actin analog in a locomoting keratocyte fragment
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Figure 4 (84 K) - Dynamics of microinjected myosin II analog in (a), locomoting and (b), stationary fragments
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Figure 5 (43 K) - Induction of fragment motility by mechanical stimulation
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Figure 6 (175 K) - Cytoskeletal rearrangement in keratocyte fragments during polarization
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Figure 7 (30 K) - Myosin dynamics in a fragment during polarization
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Figure 8 (125 K) - Model for polarization and propagation of directional locomotion
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Sequence 1 (1.8 M) - Corresponds to Figure 1b. Low magnification view of a field containing both freely locomoting keratocytes and an edge of epithelioid keratocyte colony. After addition of staurosporine (indicated) keratocytes developed numerous lamellar fragments, most of them still connected to the main cell bodies through long stalks.
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Sequence 2 (192 K) - Corresponds to Figure 3. Dynamics of speckles of injected fluorescent actin analog in a locomoting keratocyte fragment. At the leading edge, speckles are stationary with respect to the substratum and gradually decrease in brightness. At the rear, actin speckles move forward with the rear edge.
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Sequence 3 (1 M) - Corresponds to Figure 4a. Dynamics of microinjected myosin II analog in a locomoting keratocyte fragment. Myosin spots appear and increase in intensity in the lamellum, while remaining stationary with respect to the substratum. In the vicinity of the rear edge, myosin spots move forward and condense into a bundle suggesting contraction of actin-myosin II network. As the fragment travels several lengths of its body, myosin spots continuously appear in the lamellum indicating rapid turnover of myosin II polymer.
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Sequence 4 (480 K) - Corresponds to Figure 4b. Dynamics of microinjected myosin II analog in a stationary keratocyte fragment. Myosin spots move mostly centripetally but also exhibit some irregular motion and random changes in intensity.
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Sequence 5 (2.3 M) - Corresponds to Figure 5. Stationary keratocyte fragment was stimulated by a media stream from a wide micropipette tip. As a result of stimulus, the fragment polarized and started to move. Motility was persistent after the withdrawal of stimulus.
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Sequence 6 (1.3 M) - Corresponds to Figure 7. Myosin II dynamics in an initially stationary keratocyte fragment which polarized after cutting of its stalk with glass microneedle (just before the start of the sequence). Contraction of one edge of the fragment was accompanied by accumulation of myosin II, while protrusion at the opposite edge was initially free of myosin II spots.
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