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Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility.

Fischer RS, Fritz-Six KL, Fowler VM - J. Cell Biol. (2003)

Bottom Line: A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells.Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed.Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CB163, La Jolla, CA 92037, USA.

ABSTRACT
Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.

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Analyses of free barbed ends in HMEC-1 cells by rhodamine–actin incorporation in situ. (A and B) Control or GFP–Tmod3-expressing HMEC-1 cells permeabilized and labeled with rhodamine–actin. (C) Representative line scans of rhodamine–actin/fluorescent phallacidin ratio at the edge of the cell. Traces were taken from outside cell (near y axis) to inside cell (representative regions of line scans shown as white bars in A and B). See Table I for quantitation of ratiometric data from whole lamellipodia.
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fig7: Analyses of free barbed ends in HMEC-1 cells by rhodamine–actin incorporation in situ. (A and B) Control or GFP–Tmod3-expressing HMEC-1 cells permeabilized and labeled with rhodamine–actin. (C) Representative line scans of rhodamine–actin/fluorescent phallacidin ratio at the edge of the cell. Traces were taken from outside cell (near y axis) to inside cell (representative regions of line scans shown as white bars in A and B). See Table I for quantitation of ratiometric data from whole lamellipodia.

Mentions: Given the migration and polarization defects exhibited by the GFP–Tmod3-overexpressing cells, we analyzed the localization and relative levels of free barbed ends in these cells compared with control cells. When cells are permeabilized and incubated with labeled actin monomers at concentrations just above the barbed-end critical concentration, polymerization in situ from available barbed ends can be visualized (see Materials and methods; Symons and Mitchison, 1991). Incorporation of rhodamine–ATP-actin in permeabilized HMEC-1 cells demonstrates that free barbed ends are located throughout the cell, but that the most prominent population occurs at cell edges (Fig. 7 A), as expected from previous studies (Symons and Mitchison, 1991). In cells overexpressing GFP–Tmod3, this distribution is altered such that the width and intensity at cell edges are dramatically decreased (Fig. 7 B). The observed decrease in free barbed ends is not simply due to the decrease in lamellipodial F-actin (Table I), because the ratio of free barbed ends to F-actin also displays a decrease in intensity and width at the cell edge. This is demonstrated clearly by quantitative line scans (along radial lines perpendicular to the membrane) performed on ratiometric images (Fig. 7 C). Furthermore, quantitation of the ratio of free barbed ends to F-actin in whole lamellipodia indicates that cells overexpressing Tmod3 exhibit a twofold decrease in free barbed ends per relative unit of filamentous actin when compared with cells expressing endogenous levels of Tmod3 (Table I). In contrast, when the total amount of rhodamine–actin incorporation per cell is measured as a function of total cell area (to eliminate the effects of cell size and spreading), only an ∼16% decrease is observed between control cells and GFP–Tmod3-overexpressing cells (unpublished data). These data indicate that changes in free barbed ends upon overexpression of GFP–Tmod3 are confined to the cell periphery.


Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility.

Fischer RS, Fritz-Six KL, Fowler VM - J. Cell Biol. (2003)

Analyses of free barbed ends in HMEC-1 cells by rhodamine–actin incorporation in situ. (A and B) Control or GFP–Tmod3-expressing HMEC-1 cells permeabilized and labeled with rhodamine–actin. (C) Representative line scans of rhodamine–actin/fluorescent phallacidin ratio at the edge of the cell. Traces were taken from outside cell (near y axis) to inside cell (representative regions of line scans shown as white bars in A and B). See Table I for quantitation of ratiometric data from whole lamellipodia.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172920&req=5

fig7: Analyses of free barbed ends in HMEC-1 cells by rhodamine–actin incorporation in situ. (A and B) Control or GFP–Tmod3-expressing HMEC-1 cells permeabilized and labeled with rhodamine–actin. (C) Representative line scans of rhodamine–actin/fluorescent phallacidin ratio at the edge of the cell. Traces were taken from outside cell (near y axis) to inside cell (representative regions of line scans shown as white bars in A and B). See Table I for quantitation of ratiometric data from whole lamellipodia.
Mentions: Given the migration and polarization defects exhibited by the GFP–Tmod3-overexpressing cells, we analyzed the localization and relative levels of free barbed ends in these cells compared with control cells. When cells are permeabilized and incubated with labeled actin monomers at concentrations just above the barbed-end critical concentration, polymerization in situ from available barbed ends can be visualized (see Materials and methods; Symons and Mitchison, 1991). Incorporation of rhodamine–ATP-actin in permeabilized HMEC-1 cells demonstrates that free barbed ends are located throughout the cell, but that the most prominent population occurs at cell edges (Fig. 7 A), as expected from previous studies (Symons and Mitchison, 1991). In cells overexpressing GFP–Tmod3, this distribution is altered such that the width and intensity at cell edges are dramatically decreased (Fig. 7 B). The observed decrease in free barbed ends is not simply due to the decrease in lamellipodial F-actin (Table I), because the ratio of free barbed ends to F-actin also displays a decrease in intensity and width at the cell edge. This is demonstrated clearly by quantitative line scans (along radial lines perpendicular to the membrane) performed on ratiometric images (Fig. 7 C). Furthermore, quantitation of the ratio of free barbed ends to F-actin in whole lamellipodia indicates that cells overexpressing Tmod3 exhibit a twofold decrease in free barbed ends per relative unit of filamentous actin when compared with cells expressing endogenous levels of Tmod3 (Table I). In contrast, when the total amount of rhodamine–actin incorporation per cell is measured as a function of total cell area (to eliminate the effects of cell size and spreading), only an ∼16% decrease is observed between control cells and GFP–Tmod3-overexpressing cells (unpublished data). These data indicate that changes in free barbed ends upon overexpression of GFP–Tmod3 are confined to the cell periphery.

Bottom Line: A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells.Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed.Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CB163, La Jolla, CA 92037, USA.

ABSTRACT
Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.

Show MeSH