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Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.

Yamamoto M, Hilgemann DH, Feng S, Bito H, Ishihara H, Shibasaki Y, Yin HL - J. Cell Biol. (2001)

Bottom Line: However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis.PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin.Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

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PIP5KI-induced stress-fiber formation is blocked by C3. CV1 cells were microinjected with purified C3 and fluorescein-IgG as an injection marker. After 30 min, cells were fixed, permeabilized, and stained with rhodamine-phalloidin. (A) Pairwise images of rhodamine-phalloidin and fluorescein-IgG staining of two PIP5KI-overexpressing cells. The cell on the left was microinjected with C3 and fluorescein-IgG (arrow), and the one on the right was not injected. Scale bar, 40 μm. (B) Quantitation of the effect of C3 on stress-fiber formation and PDGF-induced membrane ruffling. Cells were microinjected with either C3 or fluorescein-IgG or with fluorescein-IgG alone (control). 60–80 microinjected cells were counted in each category, and the percentage of cells with thick stress fibers (in serum-starved conditions) or ruffles (after PDGF treatment) was indicated on the y axis. Data shown is from a single microinjection experiment, which is representative of two experiments.
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Figure 3: PIP5KI-induced stress-fiber formation is blocked by C3. CV1 cells were microinjected with purified C3 and fluorescein-IgG as an injection marker. After 30 min, cells were fixed, permeabilized, and stained with rhodamine-phalloidin. (A) Pairwise images of rhodamine-phalloidin and fluorescein-IgG staining of two PIP5KI-overexpressing cells. The cell on the left was microinjected with C3 and fluorescein-IgG (arrow), and the one on the right was not injected. Scale bar, 40 μm. (B) Quantitation of the effect of C3 on stress-fiber formation and PDGF-induced membrane ruffling. Cells were microinjected with either C3 or fluorescein-IgG or with fluorescein-IgG alone (control). 60–80 microinjected cells were counted in each category, and the percentage of cells with thick stress fibers (in serum-starved conditions) or ruffles (after PDGF treatment) was indicated on the y axis. Data shown is from a single microinjection experiment, which is representative of two experiments.

Mentions: We used inhibitors to examine how Rho may be involved in the PIP5KI-induced actin phenotype. Microinjected C3 decreased stress-fiber formation in PIP5KI-overexpressing cells significantly, establishing that Rho is involved. C3 also restored membrane ruffling (Fig. 3a and Fig. b). Since C3 did not stimulate ruffling of β-gal cells, we conclude that membrane ruffling was inhibited as a result of excess stress-fiber formation.


Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.

Yamamoto M, Hilgemann DH, Feng S, Bito H, Ishihara H, Shibasaki Y, Yin HL - J. Cell Biol. (2001)

PIP5KI-induced stress-fiber formation is blocked by C3. CV1 cells were microinjected with purified C3 and fluorescein-IgG as an injection marker. After 30 min, cells were fixed, permeabilized, and stained with rhodamine-phalloidin. (A) Pairwise images of rhodamine-phalloidin and fluorescein-IgG staining of two PIP5KI-overexpressing cells. The cell on the left was microinjected with C3 and fluorescein-IgG (arrow), and the one on the right was not injected. Scale bar, 40 μm. (B) Quantitation of the effect of C3 on stress-fiber formation and PDGF-induced membrane ruffling. Cells were microinjected with either C3 or fluorescein-IgG or with fluorescein-IgG alone (control). 60–80 microinjected cells were counted in each category, and the percentage of cells with thick stress fibers (in serum-starved conditions) or ruffles (after PDGF treatment) was indicated on the y axis. Data shown is from a single microinjection experiment, which is representative of two experiments.
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Related In: Results  -  Collection

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Figure 3: PIP5KI-induced stress-fiber formation is blocked by C3. CV1 cells were microinjected with purified C3 and fluorescein-IgG as an injection marker. After 30 min, cells were fixed, permeabilized, and stained with rhodamine-phalloidin. (A) Pairwise images of rhodamine-phalloidin and fluorescein-IgG staining of two PIP5KI-overexpressing cells. The cell on the left was microinjected with C3 and fluorescein-IgG (arrow), and the one on the right was not injected. Scale bar, 40 μm. (B) Quantitation of the effect of C3 on stress-fiber formation and PDGF-induced membrane ruffling. Cells were microinjected with either C3 or fluorescein-IgG or with fluorescein-IgG alone (control). 60–80 microinjected cells were counted in each category, and the percentage of cells with thick stress fibers (in serum-starved conditions) or ruffles (after PDGF treatment) was indicated on the y axis. Data shown is from a single microinjection experiment, which is representative of two experiments.
Mentions: We used inhibitors to examine how Rho may be involved in the PIP5KI-induced actin phenotype. Microinjected C3 decreased stress-fiber formation in PIP5KI-overexpressing cells significantly, establishing that Rho is involved. C3 also restored membrane ruffling (Fig. 3a and Fig. b). Since C3 did not stimulate ruffling of β-gal cells, we conclude that membrane ruffling was inhibited as a result of excess stress-fiber formation.

Bottom Line: However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis.PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin.Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

Show MeSH
Related in: MedlinePlus