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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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Overexpression of GFP–Tmod3 and fractionation of HMEC-1 cells. (A) Immunoblot of total cell lysate of cells transiently expressing either GFP or GFP–Tmod. For both endogenous Tmod3 and GFP–Tmod3, some proteolysis was observed. When GFP–Tmod is overexpressed, an apparent decrease in the endogenous Tmod3 is observed, but overall levels of Tmod3 are increased approximately sixfold. (B) Representative experiment showing [125I]protein A–probed immunoblot quantitation of Tmod3 and actin in cells overexpressing GFP or GFP–Tmod3. cpm, 125I counts per minute (detection of anti-Tmod3 anti-actin antibodies). Dark bars, Triton-insoluble fraction; light bars, Triton-soluble fraction. For GFP–Tmod3 cells, data shown are the total of both GFP–Tmod (arrowhead) and endogenous Tmod (*) bands in a given fraction.
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fig6: Overexpression of GFP–Tmod3 and fractionation of HMEC-1 cells. (A) Immunoblot of total cell lysate of cells transiently expressing either GFP or GFP–Tmod. For both endogenous Tmod3 and GFP–Tmod3, some proteolysis was observed. When GFP–Tmod is overexpressed, an apparent decrease in the endogenous Tmod3 is observed, but overall levels of Tmod3 are increased approximately sixfold. (B) Representative experiment showing [125I]protein A–probed immunoblot quantitation of Tmod3 and actin in cells overexpressing GFP or GFP–Tmod3. cpm, 125I counts per minute (detection of anti-Tmod3 anti-actin antibodies). Dark bars, Triton-insoluble fraction; light bars, Triton-soluble fraction. For GFP–Tmod3 cells, data shown are the total of both GFP–Tmod (arrowhead) and endogenous Tmod (*) bands in a given fraction.

Mentions: Because the adenovirus (Ad)-mediated expression of GFP–Tmod3 results in consistent overexpression in virtually all cells in an experiment, we were able to use a biochemical approach to compare the relative level of Tmod3 expression in both control and overexpressing cells by quantitative immunoblot analyses. This showed that overexpression of GFP–Tmod3 results in approximately a fivefold increase in total Tmod3 expression (Fig. 6, A and B) . Fractionation studies using Triton X-100 revealed that ∼30–40% of endogenous Tmod3 was associated with the cytoskeleton in control cells (Fig. 6 B). When GFP–Tmod3 was overexpressed, a five- to sixfold increase in total cytoskeleton-associated Tmod3 was observed (Fig. 6 B), with a proportional increase in the soluble pool as well. These data are consistent with the relative decrease observed in free pointed ends in the cell (Fig. 5 F). Interestingly, the overall distribution (soluble versus insoluble) of Tmod3 in overexpressing cells was not substantially different from control cells.


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

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

Overexpression of GFP–Tmod3 and fractionation of HMEC-1 cells. (A) Immunoblot of total cell lysate of cells transiently expressing either GFP or GFP–Tmod. For both endogenous Tmod3 and GFP–Tmod3, some proteolysis was observed. When GFP–Tmod is overexpressed, an apparent decrease in the endogenous Tmod3 is observed, but overall levels of Tmod3 are increased approximately sixfold. (B) Representative experiment showing [125I]protein A–probed immunoblot quantitation of Tmod3 and actin in cells overexpressing GFP or GFP–Tmod3. cpm, 125I counts per minute (detection of anti-Tmod3 anti-actin antibodies). Dark bars, Triton-insoluble fraction; light bars, Triton-soluble fraction. For GFP–Tmod3 cells, data shown are the total of both GFP–Tmod (arrowhead) and endogenous Tmod (*) bands in a given fraction.
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Related In: Results  -  Collection

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fig6: Overexpression of GFP–Tmod3 and fractionation of HMEC-1 cells. (A) Immunoblot of total cell lysate of cells transiently expressing either GFP or GFP–Tmod. For both endogenous Tmod3 and GFP–Tmod3, some proteolysis was observed. When GFP–Tmod is overexpressed, an apparent decrease in the endogenous Tmod3 is observed, but overall levels of Tmod3 are increased approximately sixfold. (B) Representative experiment showing [125I]protein A–probed immunoblot quantitation of Tmod3 and actin in cells overexpressing GFP or GFP–Tmod3. cpm, 125I counts per minute (detection of anti-Tmod3 anti-actin antibodies). Dark bars, Triton-insoluble fraction; light bars, Triton-soluble fraction. For GFP–Tmod3 cells, data shown are the total of both GFP–Tmod (arrowhead) and endogenous Tmod (*) bands in a given fraction.
Mentions: Because the adenovirus (Ad)-mediated expression of GFP–Tmod3 results in consistent overexpression in virtually all cells in an experiment, we were able to use a biochemical approach to compare the relative level of Tmod3 expression in both control and overexpressing cells by quantitative immunoblot analyses. This showed that overexpression of GFP–Tmod3 results in approximately a fivefold increase in total Tmod3 expression (Fig. 6, A and B) . Fractionation studies using Triton X-100 revealed that ∼30–40% of endogenous Tmod3 was associated with the cytoskeleton in control cells (Fig. 6 B). When GFP–Tmod3 was overexpressed, a five- to sixfold increase in total cytoskeleton-associated Tmod3 was observed (Fig. 6 B), with a proportional increase in the soluble pool as well. These data are consistent with the relative decrease observed in free pointed ends in the cell (Fig. 5 F). Interestingly, the overall distribution (soluble versus insoluble) of Tmod3 in overexpressing cells was not substantially different from control 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