While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin-myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brush-like manner near the leading edge. Shorter actin filaments often formed T-junctions with longer filaments in the brush-like area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. Polarity of actin filaments was almost uniform with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia which increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells but exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary where they became compressed and aligned resulting in formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.
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Cover Figure (224K) - Cytoskeletal organization of fish epidermal keratocytes as revealed by fluorescence and electron microscopy
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Figure 1 (288K) - Localization of actin and myosin II in keratocytes by fluorescence microscopy
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Figure 2 (288K) - Organization of actin filaments in keratocyte lamellipodia
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Figure 3 (192K) - Organization of actin filaments in the lamellipodia/cell body transition zone
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Figure 4 (288K) - Polarity of actin filaments in keratocyte cytoskeleton
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Figure 5 (64K) - Quantitation of actin filament polarity in keratocyte lamellipodia
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Figure 6 (256K) - Organization of myosin II filaments in keratocytes
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Figure 7 (128K) - Relative distribution of actin and myosin II filaments in keratocyte lamellipodia
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Figure 8 (384K) - Relative distribution of actin and myosin II filaments in the keratocyte lamellipodia-cell body transition zone
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Figure 9 (224K) - Myosin spots are stationary in the lamellipodia of a locomoting keratocyte (a), but exhibit retrograde flow in the lamellipodia of a tethered cell (b)
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Figure 10 (256K) - Formation of myosin bundles in the lamellipodia/cell body transition zone of a locomoting keratocyte is associated with forward translocation of myosin features
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Figure 11 (128K) - Models of cell body translocation
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Sequence 1 (1 M) - Dynamics of microinjected tetramethylrhodamine-myosin II in a locomoting keratocyte. New myosin spots (clusters of bipolar minifilaments) continuously form in the lamellipodium and are stationary with respect to the substratum. Myosin structures at the lamellipodium/cell body transition zone and within the cell body are obscured by bright diffuse fluorescence of soluble myosin. Frames were taken at 10 second intervals (whole sequence corresponds to 10 minutes real time), field of view approximately 90 by 50 µm.
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Sequence 2 (1 M) - Similar to sequence 1 but taken at a higher magnification. Position of the microscopic stage was changed in the middle of the sequence as keratocyte was migrating out of the field of view. In addition to dynamics in the lamellipodium, the condensation of myosin spots into fibers at the lamellipodium/cell body transition zone and forward movement of the fibers are apparent in this cell. Frames were taken at 12 second intervals (whole sequence corresponds to 6 minutes real time), field of view approximately 55 by 55 µm.
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Sequence 3 (1.9 M) - Dynamics of microinjected tetramethylrhodamine-myosin II in a keratocyte tethered at the edge of an epithelioid colony. The cell was slowly advancing with the edge of the colony. Myosin spots formed in the lamellipodium, moved backwards and condensed into fibers at the cell body boundary, while fibers in the cell body moved forward. Frames were taken at 10 second intervals (whole sequence corresponds to 6 minutes 10 seconds real time), field of view approximately 45 by 45 µm. |
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Sequence 4 (736 K) - Dynamics of myosin II at the lamellipodium/cell body transition zone of a locomoting keratocyte at high magnification. Contraction in the transition zone results in forward movement of myosin features and formation of transverse fibers. Transition zone moves forward itself as contraction sequentially involves new regions of the lamellipodium. Frames were taken at 12 second intervals (whole sequence corresponds to 2 minutes real time), field of view approximately 20 by 25 µm. |
