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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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Relative distribution of actin and myosin II filaments in the keratocyte lamellipodia–cell body transition zone. EM of a detergent-extracted cell (overview in inset) after myosin immunogold labeling shows myosin filament clusters and the boundary bundle (bottom) within an actin filament network. Actin filaments forming small bundles and changing their course can be seen at sites of myosin  localization. For better visualization, gold particles are digitally colorized in yellow. Bars, 0.2 μm.
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Figure 8: Relative distribution of actin and myosin II filaments in the keratocyte lamellipodia–cell body transition zone. EM of a detergent-extracted cell (overview in inset) after myosin immunogold labeling shows myosin filament clusters and the boundary bundle (bottom) within an actin filament network. Actin filaments forming small bundles and changing their course can be seen at sites of myosin localization. For better visualization, gold particles are digitally colorized in yellow. Bars, 0.2 μm.

Mentions: Actin and myosin filament organization, when studied separately, displayed similar patterns of rearrangement, suggesting that these events may be interdependent. To correlate the organization of actin and myosin in the same cells, we performed myosin immunogold labeling of intact cytoskeletons not treated with gelsolin (Figs. 7 and 8). Myosin staining was usually absent from the peripheral brushlike zone of lamellipodia. Individual myosin filaments that looked like rod-shaped groups of gold particles with a characteristic length of 0.4 μm were found in distal parts of lamellipodia behind the brushlike zone (Fig. 7). Clusters of myosin filaments were scattered within the actin network in the central lamellar region (Fig. 8). In the vicinity of small myosin clusters in lamellipodia, several actin filaments often seemed to converge to each myosin filament and align with it (Figs. 7 and 8), suggesting a role for myosin in the reorientation of actin filaments. A more pronounced reorientation of actin filaments at sites of myosin localization was observed in the transitional zone. Myosin filament clusters here were often found at sites where many actin filaments changed their course and converged into small bundles and asters. Myosin was highly concentrated in arc-shaped actin bundles, and individual myosin filaments that sometimes could be resolved there were mostly oriented along the bundle. Numerous gold particles were also found in the cell body (not shown). Thus, simultaneous analysis of actin and myosin organization in the same cells suggests that myosin assemblies drive the reorientation of actin filaments. Large myosin assemblies close to the cell body boundary seem to cause significant changes in the adjacent actin filament network, while individual filaments and clusters scattered in lamellipodia at most are able to align a few nearby actin filaments.


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)

Relative distribution of actin and myosin II filaments in the keratocyte lamellipodia–cell body transition zone. EM of a detergent-extracted cell (overview in inset) after myosin immunogold labeling shows myosin filament clusters and the boundary bundle (bottom) within an actin filament network. Actin filaments forming small bundles and changing their course can be seen at sites of myosin  localization. For better visualization, gold particles are digitally colorized in yellow. Bars, 0.2 μm.
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Related In: Results  -  Collection

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Figure 8: Relative distribution of actin and myosin II filaments in the keratocyte lamellipodia–cell body transition zone. EM of a detergent-extracted cell (overview in inset) after myosin immunogold labeling shows myosin filament clusters and the boundary bundle (bottom) within an actin filament network. Actin filaments forming small bundles and changing their course can be seen at sites of myosin localization. For better visualization, gold particles are digitally colorized in yellow. Bars, 0.2 μm.
Mentions: Actin and myosin filament organization, when studied separately, displayed similar patterns of rearrangement, suggesting that these events may be interdependent. To correlate the organization of actin and myosin in the same cells, we performed myosin immunogold labeling of intact cytoskeletons not treated with gelsolin (Figs. 7 and 8). Myosin staining was usually absent from the peripheral brushlike zone of lamellipodia. Individual myosin filaments that looked like rod-shaped groups of gold particles with a characteristic length of 0.4 μm were found in distal parts of lamellipodia behind the brushlike zone (Fig. 7). Clusters of myosin filaments were scattered within the actin network in the central lamellar region (Fig. 8). In the vicinity of small myosin clusters in lamellipodia, several actin filaments often seemed to converge to each myosin filament and align with it (Figs. 7 and 8), suggesting a role for myosin in the reorientation of actin filaments. A more pronounced reorientation of actin filaments at sites of myosin localization was observed in the transitional zone. Myosin filament clusters here were often found at sites where many actin filaments changed their course and converged into small bundles and asters. Myosin was highly concentrated in arc-shaped actin bundles, and individual myosin filaments that sometimes could be resolved there were mostly oriented along the bundle. Numerous gold particles were also found in the cell body (not shown). Thus, simultaneous analysis of actin and myosin organization in the same cells suggests that myosin assemblies drive the reorientation of actin filaments. Large myosin assemblies close to the cell body boundary seem to cause significant changes in the adjacent actin filament network, while individual filaments and clusters scattered in lamellipodia at most are able to align a few nearby actin filaments.

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