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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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PDGF-induced fibroblast migration is blocked by expression of mutants interfering with the Cdc42/N-WASP pathway. (A) PDGF-induced migration of GFP transiently transfected NIH-3T3 cells was assayed in collagen VI–coated 96-well microchambers and quantitated by densitometry of the spots. Cell migration is expressed as a migration index and calculated as the x-fold increase over the negative control (unstimulated cells). Figure shows the mean of five experiments; SD is indicated. (B) Percent migration (induced by 10 ng/ml PDGF) of p110CAAX- or p85α stable transfectants compared with the migration of control stable transfectants (considered 100%). Figure shows the mean of three experiments; SD is indicated. (C) Percent migration (induced by 100 ng/ml PDGF) of cells transfected with a vector encoding GFP plus the different cDNAs (indicated), compared with the migration of control cells transfected only with cDNA encoding GFP (considered 100%). Figure shows the mean of four experiments; SD is indicated.
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Figure 9: PDGF-induced fibroblast migration is blocked by expression of mutants interfering with the Cdc42/N-WASP pathway. (A) PDGF-induced migration of GFP transiently transfected NIH-3T3 cells was assayed in collagen VI–coated 96-well microchambers and quantitated by densitometry of the spots. Cell migration is expressed as a migration index and calculated as the x-fold increase over the negative control (unstimulated cells). Figure shows the mean of five experiments; SD is indicated. (B) Percent migration (induced by 10 ng/ml PDGF) of p110CAAX- or p85α stable transfectants compared with the migration of control stable transfectants (considered 100%). Figure shows the mean of three experiments; SD is indicated. (C) Percent migration (induced by 100 ng/ml PDGF) of cells transfected with a vector encoding GFP plus the different cDNAs (indicated), compared with the migration of control cells transfected only with cDNA encoding GFP (considered 100%). Figure shows the mean of four experiments; SD is indicated.

Mentions: We subsequently performed migration assays using collagen-coated microchambers. Under optimized conditions, migration was proportional to the cell numbers and to the dose of PDGF used in the assay, inducing mobilization of ∼20–25% of the 105 input cells (at 5 h with 100 ng/ml PDGF; see Materials and Methods). Cell migration was estimated in quadruplicate by densitometry of migrating cells, and the migration index, representing the x-fold increase in cell migration observed in the presence versus the absence of chemoattractant, was calculated for each condition. Fig. 9 A shows the migration index of control NIH-3T3 cells in response to increasing PDGF doses. We subsequently compared the migration indexes of the transfected samples with those of the control cells. This comparison showed that the migration efficiency of p85α stable cell lines (expressing a two-fold increase in p85α expression, Jiménez et al. 1998) was higher than the migration efficiency of control cells, particularly at suboptimal PDGF doses (10 ng/ml; Fig. 9 B). p110-CAAX cell migration was slightly lower than that of control cells at suboptimal doses of PDGF (10 ng/ml; Fig. 9 B), but similar at 100 ng/ml PDGF (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)

PDGF-induced fibroblast migration is blocked by expression of mutants interfering with the Cdc42/N-WASP pathway. (A) PDGF-induced migration of GFP transiently transfected NIH-3T3 cells was assayed in collagen VI–coated 96-well microchambers and quantitated by densitometry of the spots. Cell migration is expressed as a migration index and calculated as the x-fold increase over the negative control (unstimulated cells). Figure shows the mean of five experiments; SD is indicated. (B) Percent migration (induced by 10 ng/ml PDGF) of p110CAAX- or p85α stable transfectants compared with the migration of control stable transfectants (considered 100%). Figure shows the mean of three experiments; SD is indicated. (C) Percent migration (induced by 100 ng/ml PDGF) of cells transfected with a vector encoding GFP plus the different cDNAs (indicated), compared with the migration of control cells transfected only with cDNA encoding GFP (considered 100%). Figure shows the mean of four experiments; SD is indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192656&req=5

Figure 9: PDGF-induced fibroblast migration is blocked by expression of mutants interfering with the Cdc42/N-WASP pathway. (A) PDGF-induced migration of GFP transiently transfected NIH-3T3 cells was assayed in collagen VI–coated 96-well microchambers and quantitated by densitometry of the spots. Cell migration is expressed as a migration index and calculated as the x-fold increase over the negative control (unstimulated cells). Figure shows the mean of five experiments; SD is indicated. (B) Percent migration (induced by 10 ng/ml PDGF) of p110CAAX- or p85α stable transfectants compared with the migration of control stable transfectants (considered 100%). Figure shows the mean of three experiments; SD is indicated. (C) Percent migration (induced by 100 ng/ml PDGF) of cells transfected with a vector encoding GFP plus the different cDNAs (indicated), compared with the migration of control cells transfected only with cDNA encoding GFP (considered 100%). Figure shows the mean of four experiments; SD is indicated.
Mentions: We subsequently performed migration assays using collagen-coated microchambers. Under optimized conditions, migration was proportional to the cell numbers and to the dose of PDGF used in the assay, inducing mobilization of ∼20–25% of the 105 input cells (at 5 h with 100 ng/ml PDGF; see Materials and Methods). Cell migration was estimated in quadruplicate by densitometry of migrating cells, and the migration index, representing the x-fold increase in cell migration observed in the presence versus the absence of chemoattractant, was calculated for each condition. Fig. 9 A shows the migration index of control NIH-3T3 cells in response to increasing PDGF doses. We subsequently compared the migration indexes of the transfected samples with those of the control cells. This comparison showed that the migration efficiency of p85α stable cell lines (expressing a two-fold increase in p85α expression, Jiménez et al. 1998) was higher than the migration efficiency of control cells, particularly at suboptimal PDGF doses (10 ng/ml; Fig. 9 B). p110-CAAX cell migration was slightly lower than that of control cells at suboptimal doses of PDGF (10 ng/ml; Fig. 9 B), but similar at 100 ng/ml PDGF (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