Limits...
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.

Show MeSH

Related in: MedlinePlus

Organization of actin filaments in keratocyte lamellipodia. EM of detergent-extracted cells. (a) Overview of a locomoting cell;  (b) actin network in lamellipodia from the leading edge (top) to the transitional zone (bottom); (c) brushlike zone at the leading edge  with numerous filament ends; (d) smooth actin filament network in the middle part of lamellipodia; (e–h), T junctions (arrowheads) between filaments at the extreme leading edge (e), within the brushlike zone (f), in the central lamellipodia (g), and close to the lateral  edge of the lamellipodia (h). The cell's leading edge is oriented upward in all panels. Boxed region in a is enlarged in b; upper and lower  boxed regions in b are enlarged in c and d, respectively. Bars: (b) 1 μm; (e–h) 50 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139803&req=5

Figure 2: Organization of actin filaments in keratocyte lamellipodia. EM of detergent-extracted cells. (a) Overview of a locomoting cell; (b) actin network in lamellipodia from the leading edge (top) to the transitional zone (bottom); (c) brushlike zone at the leading edge with numerous filament ends; (d) smooth actin filament network in the middle part of lamellipodia; (e–h), T junctions (arrowheads) between filaments at the extreme leading edge (e), within the brushlike zone (f), in the central lamellipodia (g), and close to the lateral edge of the lamellipodia (h). The cell's leading edge is oriented upward in all panels. Boxed region in a is enlarged in b; upper and lower boxed regions in b are enlarged in c and d, respectively. Bars: (b) 1 μm; (e–h) 50 nm.

Mentions: In the lamellipodia of locomoting keratocytes, actin filaments were organized into networks with the highest density at the leading edge and a gradual decrease towards the nucleus (Fig. 2). Although determination of filament length distribution was not possible because of high filament density, numerous long filaments (with length comparable to the entire width of lamellipodia) were apparent. The actin network at the leading edge was characterized by an abundance of free ends in a characteristic brushlike appearance (Fig. 2, b and c) in contrast to the more smooth network in deeper parts of the lamellipodia (Fig. 2, b and d). The abundance of actin filament ends near the leading edge was suggestive of intensive actin polymerization in this area. In accord with this suggestion, the width of the brushlike zone correlated with the magnitude of lamellipodial protrusion at the respective site. As a rule, it was maximal (1.2 ± 0.3 μm) in central, forward-facing domains of the leading edge and was less at the lateral edges. The extent of the brushlike zone was greater in cells whose shape was indicative of rapid locomotion as compared to relatively stationary cells, e.g., tethered cells in keratocyte colonies.


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)

Organization of actin filaments in keratocyte lamellipodia. EM of detergent-extracted cells. (a) Overview of a locomoting cell;  (b) actin network in lamellipodia from the leading edge (top) to the transitional zone (bottom); (c) brushlike zone at the leading edge  with numerous filament ends; (d) smooth actin filament network in the middle part of lamellipodia; (e–h), T junctions (arrowheads) between filaments at the extreme leading edge (e), within the brushlike zone (f), in the central lamellipodia (g), and close to the lateral  edge of the lamellipodia (h). The cell's leading edge is oriented upward in all panels. Boxed region in a is enlarged in b; upper and lower  boxed regions in b are enlarged in c and d, respectively. Bars: (b) 1 μm; (e–h) 50 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139803&req=5

Figure 2: Organization of actin filaments in keratocyte lamellipodia. EM of detergent-extracted cells. (a) Overview of a locomoting cell; (b) actin network in lamellipodia from the leading edge (top) to the transitional zone (bottom); (c) brushlike zone at the leading edge with numerous filament ends; (d) smooth actin filament network in the middle part of lamellipodia; (e–h), T junctions (arrowheads) between filaments at the extreme leading edge (e), within the brushlike zone (f), in the central lamellipodia (g), and close to the lateral edge of the lamellipodia (h). The cell's leading edge is oriented upward in all panels. Boxed region in a is enlarged in b; upper and lower boxed regions in b are enlarged in c and d, respectively. Bars: (b) 1 μm; (e–h) 50 nm.
Mentions: In the lamellipodia of locomoting keratocytes, actin filaments were organized into networks with the highest density at the leading edge and a gradual decrease towards the nucleus (Fig. 2). Although determination of filament length distribution was not possible because of high filament density, numerous long filaments (with length comparable to the entire width of lamellipodia) were apparent. The actin network at the leading edge was characterized by an abundance of free ends in a characteristic brushlike appearance (Fig. 2, b and c) in contrast to the more smooth network in deeper parts of the lamellipodia (Fig. 2, b and d). The abundance of actin filament ends near the leading edge was suggestive of intensive actin polymerization in this area. In accord with this suggestion, the width of the brushlike zone correlated with the magnitude of lamellipodial protrusion at the respective site. As a rule, it was maximal (1.2 ± 0.3 μm) in central, forward-facing domains of the leading edge and was less at the lateral edges. The extent of the brushlike zone was greater in cells whose shape was indicative of rapid locomotion as compared to relatively stationary cells, e.g., tethered cells in keratocyte colonies.

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