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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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HMEC-1 cell morphology and migration rate are altered by transient overexpression of GFP–Tmod3. Stills of supplemental videos (available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1) of control cells (A, Video 2) or GFP–Tmod3 cells (C and D, Video 3). Bar, 10 μm. (B) Graph of average cell migration rate of control-infected or GFP–Tmod3-overexpressing HMEC-1 cells. Box and whisker plots were generated as described in the Materials and methods. n ≥ 22 cells for each measurement.
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fig4: HMEC-1 cell morphology and migration rate are altered by transient overexpression of GFP–Tmod3. Stills of supplemental videos (available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1) of control cells (A, Video 2) or GFP–Tmod3 cells (C and D, Video 3). Bar, 10 μm. (B) Graph of average cell migration rate of control-infected or GFP–Tmod3-overexpressing HMEC-1 cells. Box and whisker plots were generated as described in the Materials and methods. n ≥ 22 cells for each measurement.

Mentions: HMEC-1 cells have been shown to have an average random migration velocity of ∼10–20 μm/h, depending on the substrate (Kiosses et al., 1999). Under our conditions, HMEC-1 cells were observed to translocate at a similar rate (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1; Fig. 4, A and B) . HMEC-1 cells displayed various motile behaviors, including polarization, protrusion, contraction, and detachment (Video 2; Fig. 4 A). When adenoviral vectors were used to transiently overexpress GFP–Tmod3, HMEC-1 cells were generally less polarized in morphology and adopted a more stationary, spreading morphology (Fig. 4, C and D), although protrusion and ruffling activity at the cell periphery continued in many of these cells (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1). However, whereas only a small fraction (∼4%) of control cells failed to exhibit protrusive activity over a 2-h window, ∼22% of GFP–Tmod3-overexpressing cells failed to exhibit significant protrusive activity (see Materials and methods) during the same time period. Those cells that do transiently polarize by protruding lamellipodia often quickly lose polarity and then attempt to polarize in another direction (Video 3). Furthermore, when rates of cell translocation were measured in the overexpressing cells, the average rate was approximately half of that observed for control cells (Fig. 4 B). Although there was some variability in individual cell velocities (as demonstrated by the large data range), the average velocity difference between control cells and GFP–Tmod3-overexpressing cells was statistically significant (P ≈ 3 × 10−7). Similar effects on cell motility and morphology were also observed with viruses expressing untagged Tmod3 (unpublished data).


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

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

HMEC-1 cell morphology and migration rate are altered by transient overexpression of GFP–Tmod3. Stills of supplemental videos (available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1) of control cells (A, Video 2) or GFP–Tmod3 cells (C and D, Video 3). Bar, 10 μm. (B) Graph of average cell migration rate of control-infected or GFP–Tmod3-overexpressing HMEC-1 cells. Box and whisker plots were generated as described in the Materials and methods. n ≥ 22 cells for each measurement.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: HMEC-1 cell morphology and migration rate are altered by transient overexpression of GFP–Tmod3. Stills of supplemental videos (available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1) of control cells (A, Video 2) or GFP–Tmod3 cells (C and D, Video 3). Bar, 10 μm. (B) Graph of average cell migration rate of control-infected or GFP–Tmod3-overexpressing HMEC-1 cells. Box and whisker plots were generated as described in the Materials and methods. n ≥ 22 cells for each measurement.
Mentions: HMEC-1 cells have been shown to have an average random migration velocity of ∼10–20 μm/h, depending on the substrate (Kiosses et al., 1999). Under our conditions, HMEC-1 cells were observed to translocate at a similar rate (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1; Fig. 4, A and B) . HMEC-1 cells displayed various motile behaviors, including polarization, protrusion, contraction, and detachment (Video 2; Fig. 4 A). When adenoviral vectors were used to transiently overexpress GFP–Tmod3, HMEC-1 cells were generally less polarized in morphology and adopted a more stationary, spreading morphology (Fig. 4, C and D), although protrusion and ruffling activity at the cell periphery continued in many of these cells (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209057/DC1). However, whereas only a small fraction (∼4%) of control cells failed to exhibit protrusive activity over a 2-h window, ∼22% of GFP–Tmod3-overexpressing cells failed to exhibit significant protrusive activity (see Materials and methods) during the same time period. Those cells that do transiently polarize by protruding lamellipodia often quickly lose polarity and then attempt to polarize in another direction (Video 3). Furthermore, when rates of cell translocation were measured in the overexpressing cells, the average rate was approximately half of that observed for control cells (Fig. 4 B). Although there was some variability in individual cell velocities (as demonstrated by the large data range), the average velocity difference between control cells and GFP–Tmod3-overexpressing cells was statistically significant (P ≈ 3 × 10−7). Similar effects on cell motility and morphology were also observed with viruses expressing untagged Tmod3 (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