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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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Formation of myosin bundles in the lamellipodia–cell body transition zone  of a locomoting keratocyte is  associated with forward translocation of myosin features.  (a) An overview of a keratocyte at the start of observation  (top) and after 168 s (bottom).  Positions of two images reflect  actual displacement of the cell  in the horizontal direction.  Traces of the cell's leading and  rear edges (dashed lines) and  selected myosin features (solid  lines) are shown in the inset  with time indicated in seconds  on the vertical scale. One of  the myosin spots traced is visible on the image at time 0, others have arisen at later time  points and ended up, depending on time of their appearance and initial position, in the  lamellipodium, in the lamellipodia–cell body transition zone,  and as part of contracted myosin aggregates in the cell body  at 168 s. Traces illustrate that  myosin spots are initially stationary but become sequentially involved in forward translocation as the cell advances.  (b) Details of bundle formation in the cell region indicated  with box in a. Two myosin spots  are highlighted with red and  yellow. Dotted lines indicate  positions fixed with respect to  the substratum. Myosin spots  are compressed in a horizontal  direction (direction of locomotion), resulting in bundle formation and displacement to the  right (forward). (c) The fate of  a small myosin bundle as it  forms at the cell body boundary  (time 0) and contracts (112 s),  fragments (152–232 s), and disappears (272 s) within the cell  body. Bars, 2 μm.
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Figure 10: Formation of myosin bundles in the lamellipodia–cell body transition zone of a locomoting keratocyte is associated with forward translocation of myosin features. (a) An overview of a keratocyte at the start of observation (top) and after 168 s (bottom). Positions of two images reflect actual displacement of the cell in the horizontal direction. Traces of the cell's leading and rear edges (dashed lines) and selected myosin features (solid lines) are shown in the inset with time indicated in seconds on the vertical scale. One of the myosin spots traced is visible on the image at time 0, others have arisen at later time points and ended up, depending on time of their appearance and initial position, in the lamellipodium, in the lamellipodia–cell body transition zone, and as part of contracted myosin aggregates in the cell body at 168 s. Traces illustrate that myosin spots are initially stationary but become sequentially involved in forward translocation as the cell advances. (b) Details of bundle formation in the cell region indicated with box in a. Two myosin spots are highlighted with red and yellow. Dotted lines indicate positions fixed with respect to the substratum. Myosin spots are compressed in a horizontal direction (direction of locomotion), resulting in bundle formation and displacement to the right (forward). (c) The fate of a small myosin bundle as it forms at the cell body boundary (time 0) and contracts (112 s), fragments (152–232 s), and disappears (272 s) within 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)

Formation of myosin bundles in the lamellipodia–cell body transition zone  of a locomoting keratocyte is  associated with forward translocation of myosin features.  (a) An overview of a keratocyte at the start of observation  (top) and after 168 s (bottom).  Positions of two images reflect  actual displacement of the cell  in the horizontal direction.  Traces of the cell's leading and  rear edges (dashed lines) and  selected myosin features (solid  lines) are shown in the inset  with time indicated in seconds  on the vertical scale. One of  the myosin spots traced is visible on the image at time 0, others have arisen at later time  points and ended up, depending on time of their appearance and initial position, in the  lamellipodium, in the lamellipodia–cell body transition zone,  and as part of contracted myosin aggregates in the cell body  at 168 s. Traces illustrate that  myosin spots are initially stationary but become sequentially involved in forward translocation as the cell advances.  (b) Details of bundle formation in the cell region indicated  with box in a. Two myosin spots  are highlighted with red and  yellow. Dotted lines indicate  positions fixed with respect to  the substratum. Myosin spots  are compressed in a horizontal  direction (direction of locomotion), resulting in bundle formation and displacement to the  right (forward). (c) The fate of  a small myosin bundle as it  forms at the cell body boundary  (time 0) and contracts (112 s),  fragments (152–232 s), and disappears (272 s) within the cell  body. Bars, 2 μm.
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Figure 10: Formation of myosin bundles in the lamellipodia–cell body transition zone of a locomoting keratocyte is associated with forward translocation of myosin features. (a) An overview of a keratocyte at the start of observation (top) and after 168 s (bottom). Positions of two images reflect actual displacement of the cell in the horizontal direction. Traces of the cell's leading and rear edges (dashed lines) and selected myosin features (solid lines) are shown in the inset with time indicated in seconds on the vertical scale. One of the myosin spots traced is visible on the image at time 0, others have arisen at later time points and ended up, depending on time of their appearance and initial position, in the lamellipodium, in the lamellipodia–cell body transition zone, and as part of contracted myosin aggregates in the cell body at 168 s. Traces illustrate that myosin spots are initially stationary but become sequentially involved in forward translocation as the cell advances. (b) Details of bundle formation in the cell region indicated with box in a. Two myosin spots are highlighted with red and yellow. Dotted lines indicate positions fixed with respect to the substratum. Myosin spots are compressed in a horizontal direction (direction of locomotion), resulting in bundle formation and displacement to the right (forward). (c) The fate of a small myosin bundle as it forms at the cell body boundary (time 0) and contracts (112 s), fragments (152–232 s), and disappears (272 s) within 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