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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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Actin cytoskeleton changes induced by PDGF stimulation or p85α expression in NIH-3T3 cells cultured on collagen. NIH-3T3 cells were cultured on collagen VI–coated plates. The samples (2 × 105 NIH-3T3 cells) were transfected with different combinations of vectors encoding HA-p85α, Δ-cof-N-WASP, myc-wt-Cdc42, and myc-DN-Cdc42 (indicated). After transfection, cells were incubated in complete medium, starved, and subsequently activated as in Fig. 2. (A) Cells were fixed and stained with FITC-phalloidin or, in the case of transfected cells stained simultaneously with FITC-phalloidin (depicted) and anti-myc, anti-HA, or anti-N-WASP Ab to detect transfected cells (indicated by an arrow). (B) Paxillin staining of NIH-3T3 cells cultured on collagen and transfected, starved, and treated as in A (indicated). The figure shows a representative experiment of four performed with similar results. Bar, 100 μm.
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Figure 8: Actin cytoskeleton changes induced by PDGF stimulation or p85α expression in NIH-3T3 cells cultured on collagen. NIH-3T3 cells were cultured on collagen VI–coated plates. The samples (2 × 105 NIH-3T3 cells) were transfected with different combinations of vectors encoding HA-p85α, Δ-cof-N-WASP, myc-wt-Cdc42, and myc-DN-Cdc42 (indicated). After transfection, cells were incubated in complete medium, starved, and subsequently activated as in Fig. 2. (A) Cells were fixed and stained with FITC-phalloidin or, in the case of transfected cells stained simultaneously with FITC-phalloidin (depicted) and anti-myc, anti-HA, or anti-N-WASP Ab to detect transfected cells (indicated by an arrow). (B) Paxillin staining of NIH-3T3 cells cultured on collagen and transfected, starved, and treated as in A (indicated). The figure shows a representative experiment of four performed with similar results. Bar, 100 μm.

Mentions: We subsequently evaluated the contribution of the Cdc42/N-WASP pathway to PDGF-induced migration using collagen as a substrate. Cells cultured on collagen exhibited cytoskeletal changes (Fig. 8) similar to those observed in the absence of exogenous ECM components. In fact, p85α expression triggered an N-WASP–dependent decrease in actin stress fibers when cells were cultured on collagen (Fig. 8 A), and PDGF treatment also induced cortical actin polymerization and a decrease in LPA-induced stress fibers when cells were cultured on collagen (Fig. 8 A). Moreover, the PDGF-induced decrease in stress fibers was blocked by DN-Cdc42 or Δcof-N-WASP (Fig. 8 A), and PDGF-R stimulation also induced a Cdc42-dependent decrease in focal adhesions (Fig. 8 B). Therefore, activation of the p85/Cdc42/N-WASP pathway by PDGF induced similar cytoskeletal changes when cells were cultured on collagen or in the absence of exogenously added ECM.


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)

Actin cytoskeleton changes induced by PDGF stimulation or p85α expression in NIH-3T3 cells cultured on collagen. NIH-3T3 cells were cultured on collagen VI–coated plates. The samples (2 × 105 NIH-3T3 cells) were transfected with different combinations of vectors encoding HA-p85α, Δ-cof-N-WASP, myc-wt-Cdc42, and myc-DN-Cdc42 (indicated). After transfection, cells were incubated in complete medium, starved, and subsequently activated as in Fig. 2. (A) Cells were fixed and stained with FITC-phalloidin or, in the case of transfected cells stained simultaneously with FITC-phalloidin (depicted) and anti-myc, anti-HA, or anti-N-WASP Ab to detect transfected cells (indicated by an arrow). (B) Paxillin staining of NIH-3T3 cells cultured on collagen and transfected, starved, and treated as in A (indicated). The figure shows a representative experiment of four performed with similar results. Bar, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192656&req=5

Figure 8: Actin cytoskeleton changes induced by PDGF stimulation or p85α expression in NIH-3T3 cells cultured on collagen. NIH-3T3 cells were cultured on collagen VI–coated plates. The samples (2 × 105 NIH-3T3 cells) were transfected with different combinations of vectors encoding HA-p85α, Δ-cof-N-WASP, myc-wt-Cdc42, and myc-DN-Cdc42 (indicated). After transfection, cells were incubated in complete medium, starved, and subsequently activated as in Fig. 2. (A) Cells were fixed and stained with FITC-phalloidin or, in the case of transfected cells stained simultaneously with FITC-phalloidin (depicted) and anti-myc, anti-HA, or anti-N-WASP Ab to detect transfected cells (indicated by an arrow). (B) Paxillin staining of NIH-3T3 cells cultured on collagen and transfected, starved, and treated as in A (indicated). The figure shows a representative experiment of four performed with similar results. Bar, 100 μm.
Mentions: We subsequently evaluated the contribution of the Cdc42/N-WASP pathway to PDGF-induced migration using collagen as a substrate. Cells cultured on collagen exhibited cytoskeletal changes (Fig. 8) similar to those observed in the absence of exogenous ECM components. In fact, p85α expression triggered an N-WASP–dependent decrease in actin stress fibers when cells were cultured on collagen (Fig. 8 A), and PDGF treatment also induced cortical actin polymerization and a decrease in LPA-induced stress fibers when cells were cultured on collagen (Fig. 8 A). Moreover, the PDGF-induced decrease in stress fibers was blocked by DN-Cdc42 or Δcof-N-WASP (Fig. 8 A), and PDGF-R stimulation also induced a Cdc42-dependent decrease in focal adhesions (Fig. 8 B). Therefore, activation of the p85/Cdc42/N-WASP pathway by PDGF induced similar cytoskeletal changes when cells were cultured on collagen or in the absence of exogenously added ECM.

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