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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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Localization of Tmod3 in HMEC-1 cells. HMEC-1 cells were fixed and stained with fluorescent phallacidin and anti-Tmod3 polyclonal antibodies followed by fluorescent anti–rabbit IgG antibodies. GFP–Tmod3 colocalizes similarly with fluorescent phalloidin. Arrows indicate ruffles, arrowheads indicate small lamellipodia. In the bottom row, Tmod3 localizes to the lamellipodia, including a region behind Arp2/3. Cells expressing low levels of GFP–Tmod3 were fixed and stained with anti-p34 polyclonal antibodies. Bars, 8 μm.
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fig3: Localization of Tmod3 in HMEC-1 cells. HMEC-1 cells were fixed and stained with fluorescent phallacidin and anti-Tmod3 polyclonal antibodies followed by fluorescent anti–rabbit IgG antibodies. GFP–Tmod3 colocalizes similarly with fluorescent phalloidin. Arrows indicate ruffles, arrowheads indicate small lamellipodia. In the bottom row, Tmod3 localizes to the lamellipodia, including a region behind Arp2/3. Cells expressing low levels of GFP–Tmod3 were fixed and stained with anti-p34 polyclonal antibodies. Bars, 8 μm.

Mentions: Migrating HMEC-1 cells have small, transient lamellipodia, which often collapse into ruffles. Although these protrusions and ruffles are rich in F-actin, HMEC-1 cells also contain bundles of F-actin that are generally oriented perpendicular to the direction of movement (Fig. 3 , top, F-actin). To determine which populations of actin filaments in these cells might be targets for Tmod3 regulation, we localized endogenous Tmod3 in HMEC-1 cells by indirect immunofluorescence. In general, Tmod3 localizes preferentially to the F-actin–rich structures at the forward periphery of the cell, including lamellipodia (Fig. 3, top two rows, arrowheads) and subsequent ruffles (arrows) that form from their collapse. In contrast, Tmod3 does not localize prominently to the perpendicular F-actin bundles in the cell body. In most cells, a large diffuse staining component in the cytoplasm is also observed, likely due to fixation of a portion of the soluble pool (see below).


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

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

Localization of Tmod3 in HMEC-1 cells. HMEC-1 cells were fixed and stained with fluorescent phallacidin and anti-Tmod3 polyclonal antibodies followed by fluorescent anti–rabbit IgG antibodies. GFP–Tmod3 colocalizes similarly with fluorescent phalloidin. Arrows indicate ruffles, arrowheads indicate small lamellipodia. In the bottom row, Tmod3 localizes to the lamellipodia, including a region behind Arp2/3. Cells expressing low levels of GFP–Tmod3 were fixed and stained with anti-p34 polyclonal antibodies. Bars, 8 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172920&req=5

fig3: Localization of Tmod3 in HMEC-1 cells. HMEC-1 cells were fixed and stained with fluorescent phallacidin and anti-Tmod3 polyclonal antibodies followed by fluorescent anti–rabbit IgG antibodies. GFP–Tmod3 colocalizes similarly with fluorescent phalloidin. Arrows indicate ruffles, arrowheads indicate small lamellipodia. In the bottom row, Tmod3 localizes to the lamellipodia, including a region behind Arp2/3. Cells expressing low levels of GFP–Tmod3 were fixed and stained with anti-p34 polyclonal antibodies. Bars, 8 μm.
Mentions: Migrating HMEC-1 cells have small, transient lamellipodia, which often collapse into ruffles. Although these protrusions and ruffles are rich in F-actin, HMEC-1 cells also contain bundles of F-actin that are generally oriented perpendicular to the direction of movement (Fig. 3 , top, F-actin). To determine which populations of actin filaments in these cells might be targets for Tmod3 regulation, we localized endogenous Tmod3 in HMEC-1 cells by indirect immunofluorescence. In general, Tmod3 localizes preferentially to the F-actin–rich structures at the forward periphery of the cell, including lamellipodia (Fig. 3, top two rows, arrowheads) and subsequent ruffles (arrows) that form from their collapse. In contrast, Tmod3 does not localize prominently to the perpendicular F-actin bundles in the cell body. In most cells, a large diffuse staining component in the cytoplasm is also observed, likely due to fixation of a portion of the soluble pool (see below).

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