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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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Localization of actin and myosin II in keratocytes by fluorescence microscopy. Actin (cyan) and myosin (red) distributions are  revealed by TRITC-phalloidin and indirect immunofluorescence staining, respectively. Overall actin and myosin II organization in a  typical wing-shaped locomoting cell (a), enlarged portion of another cell exhibiting various patterns of actin and myosin mutual arrangement (b), locomoting cell of a symmetrical shape (c), and a tethered cell (d) are shown. All cells exhibit discrete myosin spots among  continuous actin network in lamellipodia, as well as accumulation of both actin and myosin at the lamellipodia/cell body boundary. Intensity profiles of actin (cyan) and myosin (red) within the cell area indicated in the “merge” panel of a are shown in the inset, and they  illustrate reverse gradients of actin and myosin in lamellipodia. (b) Examples of a myosin spot in the lamellipodia that does not coincide  with any discrete actin structure (arrowhead), myosin spots coinciding with small actin bundles merging to boundary bundles (small arrow), and colocalization of actin and myosin in the boundary bundle (large arrow). Bars, 2 μm.
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Figure 1: Localization of actin and myosin II in keratocytes by fluorescence microscopy. Actin (cyan) and myosin (red) distributions are revealed by TRITC-phalloidin and indirect immunofluorescence staining, respectively. Overall actin and myosin II organization in a typical wing-shaped locomoting cell (a), enlarged portion of another cell exhibiting various patterns of actin and myosin mutual arrangement (b), locomoting cell of a symmetrical shape (c), and a tethered cell (d) are shown. All cells exhibit discrete myosin spots among continuous actin network in lamellipodia, as well as accumulation of both actin and myosin at the lamellipodia/cell body boundary. Intensity profiles of actin (cyan) and myosin (red) within the cell area indicated in the “merge” panel of a are shown in the inset, and they illustrate reverse gradients of actin and myosin in lamellipodia. (b) Examples of a myosin spot in the lamellipodia that does not coincide with any discrete actin structure (arrowhead), myosin spots coinciding with small actin bundles merging to boundary bundles (small arrow), and colocalization of actin and myosin in the boundary bundle (large arrow). Bars, 2 μm.

Mentions: In lamellipodia, actin was organized as a continuous network often exhibiting a fine criss-cross pattern, while myosin II formed distinct spotlike accumulations (Fig. 1 a). The intensity of actin staining in lamellipodia was maximal at the leading edge and gradually decreased toward the cell body. Although it has been reported that extraction before fixation selectively depleted actin from the front of lamellipodia, resulting in an apparently uniform distribution (Small et al., 1995), cells extracted by our protocol exhibited a graded actin distribution similar to cells fixed before permeabilization. Quantitative analysis of fluorescent phalloidin binding provided a measure of the actin filament concentration and a means of evaluating extraction/fixation protocols. The ratio of actin intensity at the front of lamellipodia to that at the rear was 1.80 ± 0.40, n = 9, for cells that were first extracted and then fixed, and 1.78 ± 0.32, n = 7, for cells that were first fixed and then extracted, indicating that no significant redistribution of actin occurred upon extraction.


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)

Localization of actin and myosin II in keratocytes by fluorescence microscopy. Actin (cyan) and myosin (red) distributions are  revealed by TRITC-phalloidin and indirect immunofluorescence staining, respectively. Overall actin and myosin II organization in a  typical wing-shaped locomoting cell (a), enlarged portion of another cell exhibiting various patterns of actin and myosin mutual arrangement (b), locomoting cell of a symmetrical shape (c), and a tethered cell (d) are shown. All cells exhibit discrete myosin spots among  continuous actin network in lamellipodia, as well as accumulation of both actin and myosin at the lamellipodia/cell body boundary. Intensity profiles of actin (cyan) and myosin (red) within the cell area indicated in the “merge” panel of a are shown in the inset, and they  illustrate reverse gradients of actin and myosin in lamellipodia. (b) Examples of a myosin spot in the lamellipodia that does not coincide  with any discrete actin structure (arrowhead), myosin spots coinciding with small actin bundles merging to boundary bundles (small arrow), and colocalization of actin and myosin in the boundary bundle (large arrow). Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Localization of actin and myosin II in keratocytes by fluorescence microscopy. Actin (cyan) and myosin (red) distributions are revealed by TRITC-phalloidin and indirect immunofluorescence staining, respectively. Overall actin and myosin II organization in a typical wing-shaped locomoting cell (a), enlarged portion of another cell exhibiting various patterns of actin and myosin mutual arrangement (b), locomoting cell of a symmetrical shape (c), and a tethered cell (d) are shown. All cells exhibit discrete myosin spots among continuous actin network in lamellipodia, as well as accumulation of both actin and myosin at the lamellipodia/cell body boundary. Intensity profiles of actin (cyan) and myosin (red) within the cell area indicated in the “merge” panel of a are shown in the inset, and they illustrate reverse gradients of actin and myosin in lamellipodia. (b) Examples of a myosin spot in the lamellipodia that does not coincide with any discrete actin structure (arrowhead), myosin spots coinciding with small actin bundles merging to boundary bundles (small arrow), and colocalization of actin and myosin in the boundary bundle (large arrow). Bars, 2 μm.
Mentions: In lamellipodia, actin was organized as a continuous network often exhibiting a fine criss-cross pattern, while myosin II formed distinct spotlike accumulations (Fig. 1 a). The intensity of actin staining in lamellipodia was maximal at the leading edge and gradually decreased toward the cell body. Although it has been reported that extraction before fixation selectively depleted actin from the front of lamellipodia, resulting in an apparently uniform distribution (Small et al., 1995), cells extracted by our protocol exhibited a graded actin distribution similar to cells fixed before permeabilization. Quantitative analysis of fluorescent phalloidin binding provided a measure of the actin filament concentration and a means of evaluating extraction/fixation protocols. The ratio of actin intensity at the front of lamellipodia to that at the rear was 1.80 ± 0.40, n = 9, for cells that were first extracted and then fixed, and 1.78 ± 0.32, n = 7, for cells that were first fixed and then extracted, indicating that no significant redistribution of actin occurred upon extraction.

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