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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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Tmod3 is expressed in HMEC-1 cells. (A) Immunoblot of cell lysates or purified recombinant proteins with either anti-Tmod3 polyclonal (left) or anti-Tmod1 monoclonal (right) antibodies. Mg2+ ghosts, human erythrocyte membranes (Babcock and Fowler, 1994); Tmod1, purified recombinant human Tmod1 protein; Tmod3, purified recombinant human Tmod3 protein. (B) Quantitation of Tmod3 in HMEC-1 cells. ♦, average counts from duplicate standards of purified recombinant human Tmod3; dashed lines indicate average counts from immunoblot bands of 2 × 105 and 5 × 105 HMEC-1 cells. Line fitted to standards by least squares method was used to determine the amount of Tmod in HMEC-1 cells in the linear range of the assay.
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fig2: Tmod3 is expressed in HMEC-1 cells. (A) Immunoblot of cell lysates or purified recombinant proteins with either anti-Tmod3 polyclonal (left) or anti-Tmod1 monoclonal (right) antibodies. Mg2+ ghosts, human erythrocyte membranes (Babcock and Fowler, 1994); Tmod1, purified recombinant human Tmod1 protein; Tmod3, purified recombinant human Tmod3 protein. (B) Quantitation of Tmod3 in HMEC-1 cells. ♦, average counts from duplicate standards of purified recombinant human Tmod3; dashed lines indicate average counts from immunoblot bands of 2 × 105 and 5 × 105 HMEC-1 cells. Line fitted to standards by least squares method was used to determine the amount of Tmod in HMEC-1 cells in the linear range of the assay.

Mentions: Upon further inspection of the tissues most enriched for Tmod3 expression, we observed that these tissues were also highly vascularized. Furthermore, sections of rat spleen, lung, or intestine stained with antibodies raised against recombinant human Tmod3 display prominent staining in the cells lining blood vessels (Fig. 1 B; unpublished data) and recognize a polypeptide of ∼40 kD in immunoblots of whole spleen (Fig. 1 C). Consistent with this, a human microvascular endothelial cell line (HMEC-1) contains a 40-kD polypeptide recognized by this antibody (Fig. 2 A). As these polyclonal antibodies to Tmod3 also cross-react weakly with purified recombinant human Tmod1 protein (Fig. 2 A, left), HMEC-1 cells were probed with monoclonal antibodies specific to Tmod1 (Fig. 2 A, right). However, no Tmod1 is detected in HMEC-1 cells (Fig. 2 A, right). Given the very narrow tissue distribution of Tmod2 and Tmod4 expression in neurons and skeletal muscle, respectively (Cox and Zoghbi, 2000; Conley et al., 2001), our data suggest that Tmod3 is the only known isoform expressed in these endothelial cells.


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

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

Tmod3 is expressed in HMEC-1 cells. (A) Immunoblot of cell lysates or purified recombinant proteins with either anti-Tmod3 polyclonal (left) or anti-Tmod1 monoclonal (right) antibodies. Mg2+ ghosts, human erythrocyte membranes (Babcock and Fowler, 1994); Tmod1, purified recombinant human Tmod1 protein; Tmod3, purified recombinant human Tmod3 protein. (B) Quantitation of Tmod3 in HMEC-1 cells. ♦, average counts from duplicate standards of purified recombinant human Tmod3; dashed lines indicate average counts from immunoblot bands of 2 × 105 and 5 × 105 HMEC-1 cells. Line fitted to standards by least squares method was used to determine the amount of Tmod in HMEC-1 cells in the linear range of the assay.
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Related In: Results  -  Collection

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fig2: Tmod3 is expressed in HMEC-1 cells. (A) Immunoblot of cell lysates or purified recombinant proteins with either anti-Tmod3 polyclonal (left) or anti-Tmod1 monoclonal (right) antibodies. Mg2+ ghosts, human erythrocyte membranes (Babcock and Fowler, 1994); Tmod1, purified recombinant human Tmod1 protein; Tmod3, purified recombinant human Tmod3 protein. (B) Quantitation of Tmod3 in HMEC-1 cells. ♦, average counts from duplicate standards of purified recombinant human Tmod3; dashed lines indicate average counts from immunoblot bands of 2 × 105 and 5 × 105 HMEC-1 cells. Line fitted to standards by least squares method was used to determine the amount of Tmod in HMEC-1 cells in the linear range of the assay.
Mentions: Upon further inspection of the tissues most enriched for Tmod3 expression, we observed that these tissues were also highly vascularized. Furthermore, sections of rat spleen, lung, or intestine stained with antibodies raised against recombinant human Tmod3 display prominent staining in the cells lining blood vessels (Fig. 1 B; unpublished data) and recognize a polypeptide of ∼40 kD in immunoblots of whole spleen (Fig. 1 C). Consistent with this, a human microvascular endothelial cell line (HMEC-1) contains a 40-kD polypeptide recognized by this antibody (Fig. 2 A). As these polyclonal antibodies to Tmod3 also cross-react weakly with purified recombinant human Tmod1 protein (Fig. 2 A, left), HMEC-1 cells were probed with monoclonal antibodies specific to Tmod1 (Fig. 2 A, right). However, no Tmod1 is detected in HMEC-1 cells (Fig. 2 A, right). Given the very narrow tissue distribution of Tmod2 and Tmod4 expression in neurons and skeletal muscle, respectively (Cox and Zoghbi, 2000; Conley et al., 2001), our data suggest that Tmod3 is the only known isoform expressed in these endothelial cells.

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