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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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GFP–Tmod3 overexpression decreases Arp2/3 complex staining in lamellipodia. Control cells (A) or GFP–Tmod3- overexpressing cells (B) were stained for Arp2/3 using anti-p34 polyclonal antibodies. Arrowheads mark width of p34 staining in the lamellipodia. Bar, 10 μm.
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fig8: GFP–Tmod3 overexpression decreases Arp2/3 complex staining in lamellipodia. Control cells (A) or GFP–Tmod3- overexpressing cells (B) were stained for Arp2/3 using anti-p34 polyclonal antibodies. Arrowheads mark width of p34 staining in the lamellipodia. Bar, 10 μm.

Mentions: Previous studies using in vitro polymerization assays have shown that Tmod1 does not interact with actin filament barbed ends, nor does it bind to monomers (Weber et al., 1994, 1999). Thus, the surprising result that increased levels of Tmod3 reduced free barbed ends in the lamellipodia suggested that Tmod3 might affect other components involved in the generation of free barbed ends in lamellipodia. Using a polyclonal antibody to the p34 subunit of the Arp2/3 complex, we measured the relative levels of endogenous Arp2/3 in cell lamellipodia. In both control and GFP–Tmod3-overexpressing cells, p34 is present in the lamellipodia (Fig. 8) . However, in cells overexpressing GFP–Tmod3, the band of p34 staining was considerably less intense and narrower (Fig. 8), similar to the decrease in rhodamine–actin incorporation (Fig. 7). When the average intensity per unit area in the lamellipodia is quantitated, there is approximately threefold less Arp2/3 in the lamellipodia of GFP–Tmod3-overexpressing cells than in those of control cells (Table I). The total amount of Arp2/3 per cell is not significantly different (unpublished data). Thus, by increasing Tmod3 levels transiently in HMEC-1 cells, Arp2/3 complex appears to be displaced from the leading lamellipodia, which may result in the generation of fewer barbed ends in these structures.


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

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

GFP–Tmod3 overexpression decreases Arp2/3 complex staining in lamellipodia. Control cells (A) or GFP–Tmod3- overexpressing cells (B) were stained for Arp2/3 using anti-p34 polyclonal antibodies. Arrowheads mark width of p34 staining in the lamellipodia. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: GFP–Tmod3 overexpression decreases Arp2/3 complex staining in lamellipodia. Control cells (A) or GFP–Tmod3- overexpressing cells (B) were stained for Arp2/3 using anti-p34 polyclonal antibodies. Arrowheads mark width of p34 staining in the lamellipodia. Bar, 10 μm.
Mentions: Previous studies using in vitro polymerization assays have shown that Tmod1 does not interact with actin filament barbed ends, nor does it bind to monomers (Weber et al., 1994, 1999). Thus, the surprising result that increased levels of Tmod3 reduced free barbed ends in the lamellipodia suggested that Tmod3 might affect other components involved in the generation of free barbed ends in lamellipodia. Using a polyclonal antibody to the p34 subunit of the Arp2/3 complex, we measured the relative levels of endogenous Arp2/3 in cell lamellipodia. In both control and GFP–Tmod3-overexpressing cells, p34 is present in the lamellipodia (Fig. 8) . However, in cells overexpressing GFP–Tmod3, the band of p34 staining was considerably less intense and narrower (Fig. 8), similar to the decrease in rhodamine–actin incorporation (Fig. 7). When the average intensity per unit area in the lamellipodia is quantitated, there is approximately threefold less Arp2/3 in the lamellipodia of GFP–Tmod3-overexpressing cells than in those of control cells (Table I). The total amount of Arp2/3 per cell is not significantly different (unpublished data). Thus, by increasing Tmod3 levels transiently in HMEC-1 cells, Arp2/3 complex appears to be displaced from the leading lamellipodia, which may result in the generation of fewer barbed ends in these structures.

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