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

Myosin spots are stationary in the  lamellipodia of a locomoting keratocyte (a), but  they exhibit retrograde flow in the lamellipodia  of a tethered cell (b). General views of tetramethylrhodamine-myosin–injected cells and time-lapse sequences for boxed areas are shown with  time indicated in minute and seconds. Dotted  lines indicate fixed positions with respect to the  substratum. Selected myosin spots are shown  with arrows. In a, the marked myosin spot is stationary while in the lamellipodia, but exhibits  forward displacement at 50 s when it reaches the  cell body. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139803&req=5

Figure 9: Myosin spots are stationary in the lamellipodia of a locomoting keratocyte (a), but they exhibit retrograde flow in the lamellipodia of a tethered cell (b). General views of tetramethylrhodamine-myosin–injected cells and time-lapse sequences for boxed areas are shown with time indicated in minute and seconds. Dotted lines indicate fixed positions with respect to the substratum. Selected myosin spots are shown with arrows. In a, the marked myosin spot is stationary while in the lamellipodia, but exhibits forward displacement at 50 s when it reaches the cell body. Bars, 2 μm.

Mentions: Fluorescently labeled smooth muscle myosin was injected into both freely locomoting keratocytes and into the cells at the border of an epithelioid colony. Fluorescence microscopy of living keratocytes revealed that the distribution of injected myosin II was similar to the distribution of endogenous myosin II in extracted cells: distinct myosin spots in lamellipodia increasing in size and density towards the cell body, as well as bundles at the lamellipodia–cell body border were clearly observed (Figs. 9 and 10). The only difference was that living, microinjected cells exhibited a much brighter diffuse fluorescence in the cell body than extracted cells. This could be explained by the contribution of a soluble and extractable pool of myosin II, which (if uniformly distributed throughout the cell volume) should be more apparent in the cell body because of its thickness. Dim (compared to the cell body) diffuse fluorescence was also detected in the thin lamellipodia of living cells. This feature was used to determine the position of the cell's leading edge in fluorescence images. We conclude that, similar to fibroblasts, labeled myosin II was a faithful reporter of the distribution of endogenous myosin II.


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)

Myosin spots are stationary in the  lamellipodia of a locomoting keratocyte (a), but  they exhibit retrograde flow in the lamellipodia  of a tethered cell (b). General views of tetramethylrhodamine-myosin–injected cells and time-lapse sequences for boxed areas are shown with  time indicated in minute and seconds. Dotted  lines indicate fixed positions with respect to the  substratum. Selected myosin spots are shown  with arrows. In a, the marked myosin spot is stationary while in the lamellipodia, but exhibits  forward displacement at 50 s when it reaches the  cell body. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Myosin spots are stationary in the lamellipodia of a locomoting keratocyte (a), but they exhibit retrograde flow in the lamellipodia of a tethered cell (b). General views of tetramethylrhodamine-myosin–injected cells and time-lapse sequences for boxed areas are shown with time indicated in minute and seconds. Dotted lines indicate fixed positions with respect to the substratum. Selected myosin spots are shown with arrows. In a, the marked myosin spot is stationary while in the lamellipodia, but exhibits forward displacement at 50 s when it reaches the cell body. Bars, 2 μm.
Mentions: Fluorescently labeled smooth muscle myosin was injected into both freely locomoting keratocytes and into the cells at the border of an epithelioid colony. Fluorescence microscopy of living keratocytes revealed that the distribution of injected myosin II was similar to the distribution of endogenous myosin II in extracted cells: distinct myosin spots in lamellipodia increasing in size and density towards the cell body, as well as bundles at the lamellipodia–cell body border were clearly observed (Figs. 9 and 10). The only difference was that living, microinjected cells exhibited a much brighter diffuse fluorescence in the cell body than extracted cells. This could be explained by the contribution of a soluble and extractable pool of myosin II, which (if uniformly distributed throughout the cell volume) should be more apparent in the cell body because of its thickness. Dim (compared to the cell body) diffuse fluorescence was also detected in the thin lamellipodia of living cells. This feature was used to determine the position of the cell's leading edge in fluorescence images. We conclude that, similar to fibroblasts, labeled myosin II was a faithful reporter of the distribution of endogenous myosin II.

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