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Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation.

Svitkina TM, Verkhovsky AB, McQuade KM, Borisy GG - J. Cell Biol. (1997)

Bottom Line: Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA. tsvitkin@facstaff.wisc.edu

ABSTRACT
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 brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The 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 that 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 they 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 the 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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Polarity of actin filaments in keratocyte cytoskeleton. EM of detergent-extracted cells after myosin S1 decoration. (a) Leading  edge; (b) middle portion of a lamellipodium; (c) transitional zone; (d) boundary bundle; (e) T junctions between actin filaments at the  leading edge; (f) retraction fiber at the cell rear. Directions of pointed ends of some filaments are shown by arrowheads located next to  a filament. Filaments are oriented primarily with barbed end forward throughout the lamellipodia (a–c), while the boundary bundle has  mixed filament polarity (d). At T junctions, filaments are oriented with pointed end toward the junction (e). The retraction fiber (f) contains uniformly oriented filaments with their barbed ends to the tip of the fiber. Unlabeled intermediate filaments can be seen in some  panels. The cell's leading edge is oriented upward in all panels. Bars, 0.1 μm.
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Figure 4: Polarity of actin filaments in keratocyte cytoskeleton. EM of detergent-extracted cells after myosin S1 decoration. (a) Leading edge; (b) middle portion of a lamellipodium; (c) transitional zone; (d) boundary bundle; (e) T junctions between actin filaments at the leading edge; (f) retraction fiber at the cell rear. Directions of pointed ends of some filaments are shown by arrowheads located next to a filament. Filaments are oriented primarily with barbed end forward throughout the lamellipodia (a–c), while the boundary bundle has mixed filament polarity (d). At T junctions, filaments are oriented with pointed end toward the junction (e). The retraction fiber (f) contains uniformly oriented filaments with their barbed ends to the tip of the fiber. Unlabeled intermediate filaments can be seen in some panels. The cell's leading edge is oriented upward in all panels. Bars, 0.1 μm.

Mentions: Within the cytoskeleton, actin filament polarity is an important characteristic that determines the possible direction of myosin movement. For the determination of actin filament polarity, we used decoration with myosin S1 (Fig. 4). Free filament ends within the brushlike zone at the leading edge were identified as barbed ends. When polarity of actin filaments making T junctions was analyzed, filaments were found oriented with pointed ends toward the base of a fork (Fig. 4 e). Throughout the lamellipodia, the predominant orientation of actin filaments was with barbed ends forward (Fig. 4, a–c). Although polarity could be estimated only in a limited fraction of filaments (20–40%) because of their high density, a quantitative assay revealed a strong bias in filament polarity in lamellipodia not only at the leading edge, but in deeper parts of lamellipodia as well (Fig. 5). The fraction of filaments oriented with the pointed end forward was extremely low (∼5%) and approximately constant throughout the lamellipodia, including transitional zone. The fraction of filaments oriented with the barbed end forward was high (∼80%) and did not change appreciably with distance from the leading edge until the transitional zone was reached. Here, the fraction of filaments with the barbed end facing forward decreased with a concomitant increase in the fraction of filaments oriented approximately parallel to the leading edge, changes related to the formation of bundles. In arc-shaped bundles, the polarity of actin filaments was mixed in the center (Fig. 4 d), while the terminal parts of the bundles contained more filaments with the barbed ends facing the nearest cell edge (not shown). Retraction fibers at the rear had uniformly oriented filaments with barbed ends directed outward (Fig. 4 f). Thus, polarity of actin filaments suggests that filaments arising with the barbed ends forward at the leading edge undergo no significant reorganization throughout most of the lamellipodia; however, reorientation of filaments occurs at the lamellipodia/cell body transition.


Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation.

Svitkina TM, Verkhovsky AB, McQuade KM, Borisy GG - J. Cell Biol. (1997)

Polarity of actin filaments in keratocyte cytoskeleton. EM of detergent-extracted cells after myosin S1 decoration. (a) Leading  edge; (b) middle portion of a lamellipodium; (c) transitional zone; (d) boundary bundle; (e) T junctions between actin filaments at the  leading edge; (f) retraction fiber at the cell rear. Directions of pointed ends of some filaments are shown by arrowheads located next to  a filament. Filaments are oriented primarily with barbed end forward throughout the lamellipodia (a–c), while the boundary bundle has  mixed filament polarity (d). At T junctions, filaments are oriented with pointed end toward the junction (e). The retraction fiber (f) contains uniformly oriented filaments with their barbed ends to the tip of the fiber. Unlabeled intermediate filaments can be seen in some  panels. The cell's leading edge is oriented upward in all panels. Bars, 0.1 μm.
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Figure 4: Polarity of actin filaments in keratocyte cytoskeleton. EM of detergent-extracted cells after myosin S1 decoration. (a) Leading edge; (b) middle portion of a lamellipodium; (c) transitional zone; (d) boundary bundle; (e) T junctions between actin filaments at the leading edge; (f) retraction fiber at the cell rear. Directions of pointed ends of some filaments are shown by arrowheads located next to a filament. Filaments are oriented primarily with barbed end forward throughout the lamellipodia (a–c), while the boundary bundle has mixed filament polarity (d). At T junctions, filaments are oriented with pointed end toward the junction (e). The retraction fiber (f) contains uniformly oriented filaments with their barbed ends to the tip of the fiber. Unlabeled intermediate filaments can be seen in some panels. The cell's leading edge is oriented upward in all panels. Bars, 0.1 μm.
Mentions: Within the cytoskeleton, actin filament polarity is an important characteristic that determines the possible direction of myosin movement. For the determination of actin filament polarity, we used decoration with myosin S1 (Fig. 4). Free filament ends within the brushlike zone at the leading edge were identified as barbed ends. When polarity of actin filaments making T junctions was analyzed, filaments were found oriented with pointed ends toward the base of a fork (Fig. 4 e). Throughout the lamellipodia, the predominant orientation of actin filaments was with barbed ends forward (Fig. 4, a–c). Although polarity could be estimated only in a limited fraction of filaments (20–40%) because of their high density, a quantitative assay revealed a strong bias in filament polarity in lamellipodia not only at the leading edge, but in deeper parts of lamellipodia as well (Fig. 5). The fraction of filaments oriented with the pointed end forward was extremely low (∼5%) and approximately constant throughout the lamellipodia, including transitional zone. The fraction of filaments oriented with the barbed end forward was high (∼80%) and did not change appreciably with distance from the leading edge until the transitional zone was reached. Here, the fraction of filaments with the barbed end facing forward decreased with a concomitant increase in the fraction of filaments oriented approximately parallel to the leading edge, changes related to the formation of bundles. In arc-shaped bundles, the polarity of actin filaments was mixed in the center (Fig. 4 d), while the terminal parts of the bundles contained more filaments with the barbed ends facing the nearest cell edge (not shown). Retraction fibers at the rear had uniformly oriented filaments with barbed ends directed outward (Fig. 4 f). Thus, polarity of actin filaments suggests that filaments arising with the barbed ends forward at the leading edge undergo no significant reorganization throughout most of the lamellipodia; however, reorientation of filaments occurs at the lamellipodia/cell body transition.

Bottom Line: Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA. tsvitkin@facstaff.wisc.edu

ABSTRACT
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 brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The 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 that 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 they 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 the 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.

Show MeSH
Related in: MedlinePlus