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Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Jiménez C, Portela RA, Mellado M, Rodríguez-Frade JM, Collard J, Serrano A, Martínez-A C, Avila J, Carrera AC - J. Cell Biol. (2000)

Bottom Line: We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes.Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K.Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain.

ABSTRACT
Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

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The PDGF-induced decrease in stress fibers is blocked by dominant negative mutants of Cdc42. (A) NIH-3T3 cells (2 × 105) were transfected with cDNA encoding myc-N17-Rac (DN-Rac, 5 μg) or myc-N17-Cdc42 (N17-Cdc42, 5 μg; indicated) using calcium phosphate. Transfected cells were incubated for 16 h in medium containing 10% serum to allow exogenous protein expression, and then incubated for an additional 16-h period in medium containing 0.1% serum. Cells were stimulated as in Fig. 1, fixed, and stained with FITC-phalloidin. Transfected cells (indicated by an arrow) were detected by simultaneous indirect immunofluorescence using anti-myc primary Ab. The figure illustrates that DN-Cdc42 blocks PDGF-induced stress fiber disassembly, whereas DN-Rac inhibits PDGF-induced lamellipodial extensions, enhancing detection of PDGF-induced filopodium-like structures. (B) 2 × 105 NIH-3T3 cells were transfected with cDNA encoding GFP (2.5 μg) or with cDNA encoding GFP plus cDNA encoding DN-Rac (2.5 μg each). Cells were incubated as in A, and videos were filmed before and upon addition of PDGF (50 ng/ml) with images taken every 15 s using a Leica laser confocal microscope and TCS-NT software. The figure shows selected time points after PDGF treatment. One representative experiment of five performed with similar results is shown. Bars, 100 μm.
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Figure 2: The PDGF-induced decrease in stress fibers is blocked by dominant negative mutants of Cdc42. (A) NIH-3T3 cells (2 × 105) were transfected with cDNA encoding myc-N17-Rac (DN-Rac, 5 μg) or myc-N17-Cdc42 (N17-Cdc42, 5 μg; indicated) using calcium phosphate. Transfected cells were incubated for 16 h in medium containing 10% serum to allow exogenous protein expression, and then incubated for an additional 16-h period in medium containing 0.1% serum. Cells were stimulated as in Fig. 1, fixed, and stained with FITC-phalloidin. Transfected cells (indicated by an arrow) were detected by simultaneous indirect immunofluorescence using anti-myc primary Ab. The figure illustrates that DN-Cdc42 blocks PDGF-induced stress fiber disassembly, whereas DN-Rac inhibits PDGF-induced lamellipodial extensions, enhancing detection of PDGF-induced filopodium-like structures. (B) 2 × 105 NIH-3T3 cells were transfected with cDNA encoding GFP (2.5 μg) or with cDNA encoding GFP plus cDNA encoding DN-Rac (2.5 μg each). Cells were incubated as in A, and videos were filmed before and upon addition of PDGF (50 ng/ml) with images taken every 15 s using a Leica laser confocal microscope and TCS-NT software. The figure shows selected time points after PDGF treatment. One representative experiment of five performed with similar results is shown. Bars, 100 μm.

Mentions: Previous studies have shown that constitutive active forms of Rac or Cdc42 inhibited Rho A activation and LPA-induced (Rho A–dependent) actin stress fiber formation (Dutartre et al. 1996; Manser et al. 1997; Sander et al. 1999). We subsequently investigated whether Cdc42- or Rac-dependent signaling pathways were responsible for the inhibition of stress fibers and the adhesion complexes observed in PDGF-treated cells using dominant negative (DN) interfering mutants. Expression of DN-Rac in PDGF-treated cells partially restored actin stress fiber formation in ∼10% of the cells, although it efficiently inhibited Rac-dependent lamellipodium formation (in >95% of the cells; Fig. 2 A). In addition, expression of DN-Rac, which increases filopodia stability (Nobes and Hall 1995), revealed the formation of filopodium-like structures in the majority of the transfected cells (Fig. 2 A). In contrast, expression of DN-Cdc42 efficiently restored actin stress fiber formation in PDGF-treated cells (in 60% of the cells), and also diminished Rac-dependent membrane ruffling (in 50% of the cells; Fig. 2 A). Transfection of DN-Rac and DN-Cdc42 in these experiments yielded similar expression levels (not shown). The greater ability of DN-Cdc42 compared with DN-Rac to restore stress fiber formation in PDGF-treated cells was confirmed in microinjection experiments (data not shown).


Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Jiménez C, Portela RA, Mellado M, Rodríguez-Frade JM, Collard J, Serrano A, Martínez-A C, Avila J, Carrera AC - J. Cell Biol. (2000)

The PDGF-induced decrease in stress fibers is blocked by dominant negative mutants of Cdc42. (A) NIH-3T3 cells (2 × 105) were transfected with cDNA encoding myc-N17-Rac (DN-Rac, 5 μg) or myc-N17-Cdc42 (N17-Cdc42, 5 μg; indicated) using calcium phosphate. Transfected cells were incubated for 16 h in medium containing 10% serum to allow exogenous protein expression, and then incubated for an additional 16-h period in medium containing 0.1% serum. Cells were stimulated as in Fig. 1, fixed, and stained with FITC-phalloidin. Transfected cells (indicated by an arrow) were detected by simultaneous indirect immunofluorescence using anti-myc primary Ab. The figure illustrates that DN-Cdc42 blocks PDGF-induced stress fiber disassembly, whereas DN-Rac inhibits PDGF-induced lamellipodial extensions, enhancing detection of PDGF-induced filopodium-like structures. (B) 2 × 105 NIH-3T3 cells were transfected with cDNA encoding GFP (2.5 μg) or with cDNA encoding GFP plus cDNA encoding DN-Rac (2.5 μg each). Cells were incubated as in A, and videos were filmed before and upon addition of PDGF (50 ng/ml) with images taken every 15 s using a Leica laser confocal microscope and TCS-NT software. The figure shows selected time points after PDGF treatment. One representative experiment of five performed with similar results is shown. Bars, 100 μm.
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Related In: Results  -  Collection

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Figure 2: The PDGF-induced decrease in stress fibers is blocked by dominant negative mutants of Cdc42. (A) NIH-3T3 cells (2 × 105) were transfected with cDNA encoding myc-N17-Rac (DN-Rac, 5 μg) or myc-N17-Cdc42 (N17-Cdc42, 5 μg; indicated) using calcium phosphate. Transfected cells were incubated for 16 h in medium containing 10% serum to allow exogenous protein expression, and then incubated for an additional 16-h period in medium containing 0.1% serum. Cells were stimulated as in Fig. 1, fixed, and stained with FITC-phalloidin. Transfected cells (indicated by an arrow) were detected by simultaneous indirect immunofluorescence using anti-myc primary Ab. The figure illustrates that DN-Cdc42 blocks PDGF-induced stress fiber disassembly, whereas DN-Rac inhibits PDGF-induced lamellipodial extensions, enhancing detection of PDGF-induced filopodium-like structures. (B) 2 × 105 NIH-3T3 cells were transfected with cDNA encoding GFP (2.5 μg) or with cDNA encoding GFP plus cDNA encoding DN-Rac (2.5 μg each). Cells were incubated as in A, and videos were filmed before and upon addition of PDGF (50 ng/ml) with images taken every 15 s using a Leica laser confocal microscope and TCS-NT software. The figure shows selected time points after PDGF treatment. One representative experiment of five performed with similar results is shown. Bars, 100 μm.
Mentions: Previous studies have shown that constitutive active forms of Rac or Cdc42 inhibited Rho A activation and LPA-induced (Rho A–dependent) actin stress fiber formation (Dutartre et al. 1996; Manser et al. 1997; Sander et al. 1999). We subsequently investigated whether Cdc42- or Rac-dependent signaling pathways were responsible for the inhibition of stress fibers and the adhesion complexes observed in PDGF-treated cells using dominant negative (DN) interfering mutants. Expression of DN-Rac in PDGF-treated cells partially restored actin stress fiber formation in ∼10% of the cells, although it efficiently inhibited Rac-dependent lamellipodium formation (in >95% of the cells; Fig. 2 A). In addition, expression of DN-Rac, which increases filopodia stability (Nobes and Hall 1995), revealed the formation of filopodium-like structures in the majority of the transfected cells (Fig. 2 A). In contrast, expression of DN-Cdc42 efficiently restored actin stress fiber formation in PDGF-treated cells (in 60% of the cells), and also diminished Rac-dependent membrane ruffling (in 50% of the cells; Fig. 2 A). Transfection of DN-Rac and DN-Cdc42 in these experiments yielded similar expression levels (not shown). The greater ability of DN-Cdc42 compared with DN-Rac to restore stress fiber formation in PDGF-treated cells was confirmed in microinjection experiments (data not shown).

Bottom Line: We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes.Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K.Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain.

ABSTRACT
Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

Show MeSH
Related in: MedlinePlus