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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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Effects of decreased Tmod3 levels by siRNA(Tmod3). HMEC-1 cells were transfected with siRNA duplex oligos against Tmod3 or mock transfected. Cells were later trypsinized and seeded onto either dishes for immunoblot analyses or onto coverslips for live cell motility analyses. (A) Immunoblots of equal numbers of mock- or siRNA-transfected cells, probed with either anti-Tmod3 antibodies (top) or C4 anti-actin monoclonal antibodies (bottom). (B) Average cell migration velocities measured over 4 h on either mock-transfected (n = 72) or siRNA-transfected (n = 64) cells. Box and whisker plots were generated as described in the Materials and methods. siRNA(Tmod3)-transfected cells migrate ∼2× faster than mock cells (P < 0.001). (C) Quantitation of total free pointed ends per cell by DNase I staining. (D) Quantitation of lamellipodial free barbed ends by rhodamine–actin incorporation as a ratio of fluorescent phalloidin. siRNA(Tmod3)-transfected cells exhibit a 45% increase in free barbed ends/F in lamellipodia (n = 30 per category; P < 0.005) and a 70% increase in total free pointed ends (n = 22 per category; P = 0.0069). In C and D, standard error of the mean is indicated.
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fig9: Effects of decreased Tmod3 levels by siRNA(Tmod3). HMEC-1 cells were transfected with siRNA duplex oligos against Tmod3 or mock transfected. Cells were later trypsinized and seeded onto either dishes for immunoblot analyses or onto coverslips for live cell motility analyses. (A) Immunoblots of equal numbers of mock- or siRNA-transfected cells, probed with either anti-Tmod3 antibodies (top) or C4 anti-actin monoclonal antibodies (bottom). (B) Average cell migration velocities measured over 4 h on either mock-transfected (n = 72) or siRNA-transfected (n = 64) cells. Box and whisker plots were generated as described in the Materials and methods. siRNA(Tmod3)-transfected cells migrate ∼2× faster than mock cells (P < 0.001). (C) Quantitation of total free pointed ends per cell by DNase I staining. (D) Quantitation of lamellipodial free barbed ends by rhodamine–actin incorporation as a ratio of fluorescent phalloidin. siRNA(Tmod3)-transfected cells exhibit a 45% increase in free barbed ends/F in lamellipodia (n = 30 per category; P < 0.005) and a 70% increase in total free pointed ends (n = 22 per category; P = 0.0069). In C and D, standard error of the mean is indicated.

Mentions: To determine whether endogenous Tmod3 similarly antagonizes cell migration in HMEC-1 cells, we decreased Tmod3 expression levels using small interfering RNA (siRNA) duplexes, as previously described (Elbashir et al., 2002). Transfection of siRNA duplexes onto HMEC-1 cells decreased Tmod3 levels to ∼15% (or less) of control transfected cells (Fig. 9 A). Examination of cells by immunofluorescence staining for Tmod3 revealed that at least 70% of cells were transfected (see Materials and methods; unpublished data). Cells transfected with siRNA(Tmod3) were observed by time-lapse photomicroscopy to determine if the decrease in Tmod3 levels had an effect on cell migration rates. Indeed, these cells exhibited a significantly increased average cell velocity compared with control transfected cells (Fig. 9 B). Thus, reduced Tmod3 levels lead to the opposite effects induced by Tmod3–GFP overexpression, indicating that both endogenous and overexpressed Tmod3 exert an inhibitory effect on cell migration.


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

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

Effects of decreased Tmod3 levels by siRNA(Tmod3). HMEC-1 cells were transfected with siRNA duplex oligos against Tmod3 or mock transfected. Cells were later trypsinized and seeded onto either dishes for immunoblot analyses or onto coverslips for live cell motility analyses. (A) Immunoblots of equal numbers of mock- or siRNA-transfected cells, probed with either anti-Tmod3 antibodies (top) or C4 anti-actin monoclonal antibodies (bottom). (B) Average cell migration velocities measured over 4 h on either mock-transfected (n = 72) or siRNA-transfected (n = 64) cells. Box and whisker plots were generated as described in the Materials and methods. siRNA(Tmod3)-transfected cells migrate ∼2× faster than mock cells (P < 0.001). (C) Quantitation of total free pointed ends per cell by DNase I staining. (D) Quantitation of lamellipodial free barbed ends by rhodamine–actin incorporation as a ratio of fluorescent phalloidin. siRNA(Tmod3)-transfected cells exhibit a 45% increase in free barbed ends/F in lamellipodia (n = 30 per category; P < 0.005) and a 70% increase in total free pointed ends (n = 22 per category; P = 0.0069). In C and D, standard error of the mean is indicated.
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Related In: Results  -  Collection

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fig9: Effects of decreased Tmod3 levels by siRNA(Tmod3). HMEC-1 cells were transfected with siRNA duplex oligos against Tmod3 or mock transfected. Cells were later trypsinized and seeded onto either dishes for immunoblot analyses or onto coverslips for live cell motility analyses. (A) Immunoblots of equal numbers of mock- or siRNA-transfected cells, probed with either anti-Tmod3 antibodies (top) or C4 anti-actin monoclonal antibodies (bottom). (B) Average cell migration velocities measured over 4 h on either mock-transfected (n = 72) or siRNA-transfected (n = 64) cells. Box and whisker plots were generated as described in the Materials and methods. siRNA(Tmod3)-transfected cells migrate ∼2× faster than mock cells (P < 0.001). (C) Quantitation of total free pointed ends per cell by DNase I staining. (D) Quantitation of lamellipodial free barbed ends by rhodamine–actin incorporation as a ratio of fluorescent phalloidin. siRNA(Tmod3)-transfected cells exhibit a 45% increase in free barbed ends/F in lamellipodia (n = 30 per category; P < 0.005) and a 70% increase in total free pointed ends (n = 22 per category; P = 0.0069). In C and D, standard error of the mean is indicated.
Mentions: To determine whether endogenous Tmod3 similarly antagonizes cell migration in HMEC-1 cells, we decreased Tmod3 expression levels using small interfering RNA (siRNA) duplexes, as previously described (Elbashir et al., 2002). Transfection of siRNA duplexes onto HMEC-1 cells decreased Tmod3 levels to ∼15% (or less) of control transfected cells (Fig. 9 A). Examination of cells by immunofluorescence staining for Tmod3 revealed that at least 70% of cells were transfected (see Materials and methods; unpublished data). Cells transfected with siRNA(Tmod3) were observed by time-lapse photomicroscopy to determine if the decrease in Tmod3 levels had an effect on cell migration rates. Indeed, these cells exhibited a significantly increased average cell velocity compared with control transfected cells (Fig. 9 B). Thus, reduced Tmod3 levels lead to the opposite effects induced by Tmod3–GFP overexpression, indicating that both endogenous and overexpressed Tmod3 exert an inhibitory effect on cell migration.

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