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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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N-WASP mediates the inhibition of actin stress fibers induced by PDGF and by p85α. 2 × 105 NIH-3T3 cells were transfected as in Fig. 2 with a total of 5 μg cDNA. Different combinations of vectors encoding HA-p85α, N-WASP, Δ-cof-N-WASP, and myc-v-Cdc42 were used (indicated). (A) 50 μg of lysates from cells transfected with cDNAs encoding control vector, or N-WASP, or Δ-cof-N-WASP were resolved by SDS-PAGE, transferred onto nitrocellulose, and examined by Western blot using anti-N-WASP Ab. (B) After transfection with the indicated plasmids, cells were cultured and activated as in Fig. 2. Cells were subsequently fixed and stained simultaneously with FITC-phalloidin (depicted) and with anti-myc, anti-HA, or anti–N-WASP Ab (indirect immunofluorescence, not shown) to detect positive transfected cells (indicated by an arrow). Throughout the experiment, Δ-cof-N-WASP expression blocked stress fiber disassembly, which was induced by p85α, PDGF, or v-Cdc42. The figure illustrates one representative experiment of five performed with similar results. Bar, 100 μm.
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Figure 7: N-WASP mediates the inhibition of actin stress fibers induced by PDGF and by p85α. 2 × 105 NIH-3T3 cells were transfected as in Fig. 2 with a total of 5 μg cDNA. Different combinations of vectors encoding HA-p85α, N-WASP, Δ-cof-N-WASP, and myc-v-Cdc42 were used (indicated). (A) 50 μg of lysates from cells transfected with cDNAs encoding control vector, or N-WASP, or Δ-cof-N-WASP were resolved by SDS-PAGE, transferred onto nitrocellulose, and examined by Western blot using anti-N-WASP Ab. (B) After transfection with the indicated plasmids, cells were cultured and activated as in Fig. 2. Cells were subsequently fixed and stained simultaneously with FITC-phalloidin (depicted) and with anti-myc, anti-HA, or anti–N-WASP Ab (indirect immunofluorescence, not shown) to detect positive transfected cells (indicated by an arrow). Throughout the experiment, Δ-cof-N-WASP expression blocked stress fiber disassembly, which was induced by p85α, PDGF, or v-Cdc42. The figure illustrates one representative experiment of five performed with similar results. Bar, 100 μm.

Mentions: It has been shown that α-Pak contributes to mediating the decrease in stress fibers observed in cells expressing v-Rac or v-Cdc42 (Manser et al. 1997; Zhao et al. 1998). However, as the PDGF-induced decrease in stress fibers was, to a large extent, Cdc42-dependent, we searched for a Cdc42-specific effector that might mediate this action and we considered N-WASP. N-WASP is an effector of Cdc42 that mediates filopodia formation, a function for which the cofilin homologous region of N-WASP seems to be essential (Miki et al. 1996, Miki et al. 1998). Thus, we examined the consequences on actin stress fiber formation of transfecting wt-N-WASP or Δcof-N-WASP, which were expressed to a similar extent (Fig. 7 A). Whereas in cells expressing N-WASP, p85α mediated a pronounced decrease in actin stress fibers, Δcof-N-WASP expression restored, to a large extent, actin stress fiber formation in p85α-expressing cells (in >95%; Fig. 7 B). Similar phenotypic changes were observed when p85α and N-WASP plasmids were cotransfected with wt-Cdc42 (not shown). In contrast, expression of wt-Cdc42 alone, N-WASP alone, or Δcof-N-WASP in serum-starved cells had no significant effects on the actin cytoskeleton (our data not shown; Miki et al. 1998). Thus, N-WASP appears to be an essential mediator of the p85α-induced Cdc42-dependent decrease in actin stress fibers.


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)

N-WASP mediates the inhibition of actin stress fibers induced by PDGF and by p85α. 2 × 105 NIH-3T3 cells were transfected as in Fig. 2 with a total of 5 μg cDNA. Different combinations of vectors encoding HA-p85α, N-WASP, Δ-cof-N-WASP, and myc-v-Cdc42 were used (indicated). (A) 50 μg of lysates from cells transfected with cDNAs encoding control vector, or N-WASP, or Δ-cof-N-WASP were resolved by SDS-PAGE, transferred onto nitrocellulose, and examined by Western blot using anti-N-WASP Ab. (B) After transfection with the indicated plasmids, cells were cultured and activated as in Fig. 2. Cells were subsequently fixed and stained simultaneously with FITC-phalloidin (depicted) and with anti-myc, anti-HA, or anti–N-WASP Ab (indirect immunofluorescence, not shown) to detect positive transfected cells (indicated by an arrow). Throughout the experiment, Δ-cof-N-WASP expression blocked stress fiber disassembly, which was induced by p85α, PDGF, or v-Cdc42. The figure illustrates one representative experiment of five performed with similar results. Bar, 100 μm.
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Related In: Results  -  Collection

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Figure 7: N-WASP mediates the inhibition of actin stress fibers induced by PDGF and by p85α. 2 × 105 NIH-3T3 cells were transfected as in Fig. 2 with a total of 5 μg cDNA. Different combinations of vectors encoding HA-p85α, N-WASP, Δ-cof-N-WASP, and myc-v-Cdc42 were used (indicated). (A) 50 μg of lysates from cells transfected with cDNAs encoding control vector, or N-WASP, or Δ-cof-N-WASP were resolved by SDS-PAGE, transferred onto nitrocellulose, and examined by Western blot using anti-N-WASP Ab. (B) After transfection with the indicated plasmids, cells were cultured and activated as in Fig. 2. Cells were subsequently fixed and stained simultaneously with FITC-phalloidin (depicted) and with anti-myc, anti-HA, or anti–N-WASP Ab (indirect immunofluorescence, not shown) to detect positive transfected cells (indicated by an arrow). Throughout the experiment, Δ-cof-N-WASP expression blocked stress fiber disassembly, which was induced by p85α, PDGF, or v-Cdc42. The figure illustrates one representative experiment of five performed with similar results. Bar, 100 μm.
Mentions: It has been shown that α-Pak contributes to mediating the decrease in stress fibers observed in cells expressing v-Rac or v-Cdc42 (Manser et al. 1997; Zhao et al. 1998). However, as the PDGF-induced decrease in stress fibers was, to a large extent, Cdc42-dependent, we searched for a Cdc42-specific effector that might mediate this action and we considered N-WASP. N-WASP is an effector of Cdc42 that mediates filopodia formation, a function for which the cofilin homologous region of N-WASP seems to be essential (Miki et al. 1996, Miki et al. 1998). Thus, we examined the consequences on actin stress fiber formation of transfecting wt-N-WASP or Δcof-N-WASP, which were expressed to a similar extent (Fig. 7 A). Whereas in cells expressing N-WASP, p85α mediated a pronounced decrease in actin stress fibers, Δcof-N-WASP expression restored, to a large extent, actin stress fiber formation in p85α-expressing cells (in >95%; Fig. 7 B). Similar phenotypic changes were observed when p85α and N-WASP plasmids were cotransfected with wt-Cdc42 (not shown). In contrast, expression of wt-Cdc42 alone, N-WASP alone, or Δcof-N-WASP in serum-starved cells had no significant effects on the actin cytoskeleton (our data not shown; Miki et al. 1998). Thus, N-WASP appears to be an essential mediator of the p85α-induced Cdc42-dependent decrease in actin stress fibers.

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