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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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Effects of PIP5KI overexpression on PIP2 levels. CV1 cells were infected with recombinant β-gal, HA-tagged PIP5KI (WT, wild type) or HA-tagged PIP5KI K138A mutant adenovirus vectors, and cultured in serum-free medium. (A) 32P incorporation into phospholipids. Cells were labeled with 32P for 4 h. Lipids were extracted, resolved by TLC, and detected by autoradiography. Duplicate samples for each condition were shown. Lipids were identified by using lipid standards. (B) Lipid profiles resolved by HPLC. The elution of glycerol (g)-phospholipid standards was indicated. PS, phosphatidylserine; PI, phosphatidylinositol. Data shown are from a representative experiment repeated three times.
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Figure 1: Effects of PIP5KI overexpression on PIP2 levels. CV1 cells were infected with recombinant β-gal, HA-tagged PIP5KI (WT, wild type) or HA-tagged PIP5KI K138A mutant adenovirus vectors, and cultured in serum-free medium. (A) 32P incorporation into phospholipids. Cells were labeled with 32P for 4 h. Lipids were extracted, resolved by TLC, and detected by autoradiography. Duplicate samples for each condition were shown. Lipids were identified by using lipid standards. (B) Lipid profiles resolved by HPLC. The elution of glycerol (g)-phospholipid standards was indicated. PS, phosphatidylserine; PI, phosphatidylinositol. Data shown are from a representative experiment repeated three times.

Mentions: We used adenovirus to introduce HA-PIP5KI into cells. Immunofluorescence staining with anti–HA showed that close to 95% of the CV1 cells were infected by this procedure. The high percentage of infected cells allowed us to use biochemical assays to determine precisely how the phosphoinositide levels are changed and how individual players in the regulatory pathway are affected. Using TLC to monitor the phosphoinositide levels, we found that in PIP5KI-overexpressing cells, 32P incorporation into PIP2 and PI4P in the experiment shown in Fig. 1 A was 380 and 30%, respectively, of that of β-gal–infected cells. These results established the extent to which PIP2 level was increased, and showed that there was a reciprocal relation between PIP2 and PI4P. Although PI4P is generally assumed to be present in large excess compared with PIP2, our results suggest that the possibility that a subset of the PI4P pools may be limiting in these cells and that PIP5KI overexpression depletes this PI4P pool by converting it to PIP2.


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)

Effects of PIP5KI overexpression on PIP2 levels. CV1 cells were infected with recombinant β-gal, HA-tagged PIP5KI (WT, wild type) or HA-tagged PIP5KI K138A mutant adenovirus vectors, and cultured in serum-free medium. (A) 32P incorporation into phospholipids. Cells were labeled with 32P for 4 h. Lipids were extracted, resolved by TLC, and detected by autoradiography. Duplicate samples for each condition were shown. Lipids were identified by using lipid standards. (B) Lipid profiles resolved by HPLC. The elution of glycerol (g)-phospholipid standards was indicated. PS, phosphatidylserine; PI, phosphatidylinositol. Data shown are from a representative experiment repeated three times.
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Related In: Results  -  Collection

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Figure 1: Effects of PIP5KI overexpression on PIP2 levels. CV1 cells were infected with recombinant β-gal, HA-tagged PIP5KI (WT, wild type) or HA-tagged PIP5KI K138A mutant adenovirus vectors, and cultured in serum-free medium. (A) 32P incorporation into phospholipids. Cells were labeled with 32P for 4 h. Lipids were extracted, resolved by TLC, and detected by autoradiography. Duplicate samples for each condition were shown. Lipids were identified by using lipid standards. (B) Lipid profiles resolved by HPLC. The elution of glycerol (g)-phospholipid standards was indicated. PS, phosphatidylserine; PI, phosphatidylinositol. Data shown are from a representative experiment repeated three times.
Mentions: We used adenovirus to introduce HA-PIP5KI into cells. Immunofluorescence staining with anti–HA showed that close to 95% of the CV1 cells were infected by this procedure. The high percentage of infected cells allowed us to use biochemical assays to determine precisely how the phosphoinositide levels are changed and how individual players in the regulatory pathway are affected. Using TLC to monitor the phosphoinositide levels, we found that in PIP5KI-overexpressing cells, 32P incorporation into PIP2 and PI4P in the experiment shown in Fig. 1 A was 380 and 30%, respectively, of that of β-gal–infected cells. These results established the extent to which PIP2 level was increased, and showed that there was a reciprocal relation between PIP2 and PI4P. Although PI4P is generally assumed to be present in large excess compared with PIP2, our results suggest that the possibility that a subset of the PI4P pools may be limiting in these cells and that PIP5KI overexpression depletes this PI4P pool by converting it to PIP2.

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