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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 caps actin filament pointed ends in vivo. (A–C) Permeabilized cell expressing GFP–Tmod3 stained with DNase I or coumarin–phalloidin and DAPI. (A) Rhodamine–DNase I; (B) DAPI + phallacidin; (C) GFP–Tmod3. (D and E) Permeabilized control-infected cell stained with DNase I (D) and coumarin–phalloidin (E). DAPI staining labels nuclei in the same channel as coumarin–phalloidin. Bar, 15 μm. (F) Quantitation of nonnuclear DNase I staining in control and GFP–Tmod3-expressing cells. Values are averages from >20 cells for each cell type, standard deviation is shown by error bars.
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fig5: Tmod3 caps actin filament pointed ends in vivo. (A–C) Permeabilized cell expressing GFP–Tmod3 stained with DNase I or coumarin–phalloidin and DAPI. (A) Rhodamine–DNase I; (B) DAPI + phallacidin; (C) GFP–Tmod3. (D and E) Permeabilized control-infected cell stained with DNase I (D) and coumarin–phalloidin (E). DAPI staining labels nuclei in the same channel as coumarin–phalloidin. Bar, 15 μm. (F) Quantitation of nonnuclear DNase I staining in control and GFP–Tmod3-expressing cells. Values are averages from >20 cells for each cell type, standard deviation is shown by error bars.

Mentions: To determine whether an increase in Tmod3 leads to increased capping of existing free pointed ends in HMEC-1 cells, we used DNase I as a marker for free pointed ends in cells in situ (Chan et al., 2000). In control HMEC-1 cells, DNase I–stained free pointed ends are located throughout the cell, with a concentration at the cell periphery, both at the anterior edge and in apparent tail extensions (Fig. 5 D). As expected, DNase I also prominently stained cell nuclei, which were identified with DAPI staining (Fig. 5, B and E). In cells in which GFP–Tmod3 was overexpressed, there was a decrease in DNase I staining (Fig. 5, compare A with B) that was particularly significant in regions where GFP–Tmod3 was found (e.g., at the cell periphery; Fig. 5 C). Note that the extraction of monomers before staining with this procedure causes some cytoplasmic GFP–Tmod3 to collapse onto the nucleus (Fig. 5 C), but this is not normally observed in live or fixed cells (e.g., compare with Fig. 3). When regions of nuclear staining are excluded, the DNase I staining intensity can thus be used to quantitate the relative levels of free pointed ends in the cells. When compared with control-infected cells, cells overexpressing GFP–Tmod3 exhibit a fivefold reduction in free pointed ends (Fig. 5 F), roughly proportional to the increase in cytoskeleton-associated Tmod3 in the cells (see below). These data suggest that GFP–Tmod3 caps a large proportion of the available pointed ends in actin structures throughout the cell, including at the leading edge.


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

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

Tmod3 caps actin filament pointed ends in vivo. (A–C) Permeabilized cell expressing GFP–Tmod3 stained with DNase I or coumarin–phalloidin and DAPI. (A) Rhodamine–DNase I; (B) DAPI + phallacidin; (C) GFP–Tmod3. (D and E) Permeabilized control-infected cell stained with DNase I (D) and coumarin–phalloidin (E). DAPI staining labels nuclei in the same channel as coumarin–phalloidin. Bar, 15 μm. (F) Quantitation of nonnuclear DNase I staining in control and GFP–Tmod3-expressing cells. Values are averages from >20 cells for each cell type, standard deviation is shown by error bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172920&req=5

fig5: Tmod3 caps actin filament pointed ends in vivo. (A–C) Permeabilized cell expressing GFP–Tmod3 stained with DNase I or coumarin–phalloidin and DAPI. (A) Rhodamine–DNase I; (B) DAPI + phallacidin; (C) GFP–Tmod3. (D and E) Permeabilized control-infected cell stained with DNase I (D) and coumarin–phalloidin (E). DAPI staining labels nuclei in the same channel as coumarin–phalloidin. Bar, 15 μm. (F) Quantitation of nonnuclear DNase I staining in control and GFP–Tmod3-expressing cells. Values are averages from >20 cells for each cell type, standard deviation is shown by error bars.
Mentions: To determine whether an increase in Tmod3 leads to increased capping of existing free pointed ends in HMEC-1 cells, we used DNase I as a marker for free pointed ends in cells in situ (Chan et al., 2000). In control HMEC-1 cells, DNase I–stained free pointed ends are located throughout the cell, with a concentration at the cell periphery, both at the anterior edge and in apparent tail extensions (Fig. 5 D). As expected, DNase I also prominently stained cell nuclei, which were identified with DAPI staining (Fig. 5, B and E). In cells in which GFP–Tmod3 was overexpressed, there was a decrease in DNase I staining (Fig. 5, compare A with B) that was particularly significant in regions where GFP–Tmod3 was found (e.g., at the cell periphery; Fig. 5 C). Note that the extraction of monomers before staining with this procedure causes some cytoplasmic GFP–Tmod3 to collapse onto the nucleus (Fig. 5 C), but this is not normally observed in live or fixed cells (e.g., compare with Fig. 3). When regions of nuclear staining are excluded, the DNase I staining intensity can thus be used to quantitate the relative levels of free pointed ends in the cells. When compared with control-infected cells, cells overexpressing GFP–Tmod3 exhibit a fivefold reduction in free pointed ends (Fig. 5 F), roughly proportional to the increase in cytoskeleton-associated Tmod3 in the cells (see below). These data suggest that GFP–Tmod3 caps a large proportion of the available pointed ends in actin structures throughout the cell, including at the leading edge.

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