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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 a novel Tmod isoform expressed in endothelial cells. (A) Tmod3 caps actin filament pointed ends with a similar affinity as Tmods 1 and 4. Pointed-end capping activities of human Tmod3 (□, large dash), human Tmod1 (▴, small dash), and chicken Tmod4 (•, solid). The extent of pointed-end capping is plotted as the initial rate of elongation in the presence of Tmod, divided by the initial rate of elongation for the control actin (R/CR), versus increasing Tmod concentration, such that when R/CR = 0, 100% of pointed ends are capped. (B) Cryosection of rat spleen stained with polyclonal anti-Tmod3 antibodies. Bar, 150 μm. (C) Immunoblot of total rat spleen protein with anti-Tmod3 antibody.
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fig1: Tmod3 is a novel Tmod isoform expressed in endothelial cells. (A) Tmod3 caps actin filament pointed ends with a similar affinity as Tmods 1 and 4. Pointed-end capping activities of human Tmod3 (□, large dash), human Tmod1 (▴, small dash), and chicken Tmod4 (•, solid). The extent of pointed-end capping is plotted as the initial rate of elongation in the presence of Tmod, divided by the initial rate of elongation for the control actin (R/CR), versus increasing Tmod concentration, such that when R/CR = 0, 100% of pointed ends are capped. (B) Cryosection of rat spleen stained with polyclonal anti-Tmod3 antibodies. Bar, 150 μm. (C) Immunoblot of total rat spleen protein with anti-Tmod3 antibody.

Mentions: Previous studies have identified a family of four canonical Tmod genes in vertebrate species, based on homology to known cDNA sequences (Cox and Zoghbi, 2000; Conley et al., 2001). A new isoform identified in vertebrates was designated as Tmod3. Like other Tmod isoforms, Tmod3 inhibits elongation from the pointed ends of gelsolin-capped actin filaments in a concentration-dependent manner (Fig. 1 A). When initial rates of elongation as a function of Tmod3 concentration are analyzed by double reciprocal plots, the Kd of Tmod3 for capping pointed ends of actin filaments is ∼110 nM. This is similar to the Kds for capping pointed ends of Tmod1 (Kd ≈ 180 nM) and Tmod4 (Kd ≈ 80 nM) (Fig. 1 A; Weber et al., 1999). However, whereas other Tmod isoforms are restricted to only a few differentiated cell types, Tmod3 was found in nearly all tissues tested (Cox and Zoghbi, 2000; Conley et al., 2001; unpublished data).


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

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

Tmod3 is a novel Tmod isoform expressed in endothelial cells. (A) Tmod3 caps actin filament pointed ends with a similar affinity as Tmods 1 and 4. Pointed-end capping activities of human Tmod3 (□, large dash), human Tmod1 (▴, small dash), and chicken Tmod4 (•, solid). The extent of pointed-end capping is plotted as the initial rate of elongation in the presence of Tmod, divided by the initial rate of elongation for the control actin (R/CR), versus increasing Tmod concentration, such that when R/CR = 0, 100% of pointed ends are capped. (B) Cryosection of rat spleen stained with polyclonal anti-Tmod3 antibodies. Bar, 150 μm. (C) Immunoblot of total rat spleen protein with anti-Tmod3 antibody.
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Related In: Results  -  Collection

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

fig1: Tmod3 is a novel Tmod isoform expressed in endothelial cells. (A) Tmod3 caps actin filament pointed ends with a similar affinity as Tmods 1 and 4. Pointed-end capping activities of human Tmod3 (□, large dash), human Tmod1 (▴, small dash), and chicken Tmod4 (•, solid). The extent of pointed-end capping is plotted as the initial rate of elongation in the presence of Tmod, divided by the initial rate of elongation for the control actin (R/CR), versus increasing Tmod concentration, such that when R/CR = 0, 100% of pointed ends are capped. (B) Cryosection of rat spleen stained with polyclonal anti-Tmod3 antibodies. Bar, 150 μm. (C) Immunoblot of total rat spleen protein with anti-Tmod3 antibody.
Mentions: Previous studies have identified a family of four canonical Tmod genes in vertebrate species, based on homology to known cDNA sequences (Cox and Zoghbi, 2000; Conley et al., 2001). A new isoform identified in vertebrates was designated as Tmod3. Like other Tmod isoforms, Tmod3 inhibits elongation from the pointed ends of gelsolin-capped actin filaments in a concentration-dependent manner (Fig. 1 A). When initial rates of elongation as a function of Tmod3 concentration are analyzed by double reciprocal plots, the Kd of Tmod3 for capping pointed ends of actin filaments is ∼110 nM. This is similar to the Kds for capping pointed ends of Tmod1 (Kd ≈ 180 nM) and Tmod4 (Kd ≈ 80 nM) (Fig. 1 A; Weber et al., 1999). However, whereas other Tmod isoforms are restricted to only a few differentiated cell types, Tmod3 was found in nearly all tissues tested (Cox and Zoghbi, 2000; Conley et al., 2001; unpublished data).

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
Related in: MedlinePlus