Limits...
Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

Lyulcheva E, Taylor E, Michael M, Vehlow A, Tan S, Fletcher A, Krause M, Bennett D - Dev. Cell (2008)

Bottom Line: Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner.We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal.We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK.

ABSTRACT
MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

Show MeSH

Related in: MedlinePlus

pico Interacts with ena and Modifies Actin Dynamics(A) Representative wing discs of the indicated genotypes stained for F-actin. Ectopic expression of pico or ena results in elevated F-actin, whereas ectopic picoIR reduces F-actin. pico-induced F-actin formation is dominantly suppressed by ena210. Ectopic expression, driven by en-GAL4, is limited to the posterior compartment marked by GFP. Quantitation of ratios of anterior:posterior F-actin levels is shown at the bottom of (A) (±SD).(B) Immunoblots showing levels of total actin normalized to tubulin in extracts from wing discs of the indicated genotypes. Mean actin levels from six independent experiments are expressed below as percentage of the control ± SD.(C) Ectopic expression of Myc-pico using MS1096-GAL4 results in elevated F-actin staining in the dorsal region of the wing disc where Myc-pico levels are highest. This effect is suppressed by coexpression of DN-Egfr. Quantitation of ratios of ventral:dorsal F-actin levels (±SD) is shown in the upper right corners of the upper panels.(D) Pico full-length, but not picoRA-PH, binds the Ena EVH1 domain, but not an unrelated control (NIPP1), in the yeast two-hybrid system. Interaction is indicated by blue X-gal (X) and growth on auxotrophic media at high (H) and low (L) density.(E) Ena coimmunoprecipitates with Myc-tagged pico from MS1096 > Myc-pico wing disc extracts. Lower panel shows protein immunoblot analyses of total cell lysates to control for loading.(F) Wing images of the indicated genotypes showing functional interactions between pico and ena in the wing. Wing from ena210 heterozygote resembles wild-type. ena210 dominantly suppresses the effect of ectopic pico on tissue overgrowth; compare with the effect of one copy of pico alone. Ectopic ena drives tissue overgrowth. The effect of ectopic ena is not enhanced by coexpression of pico, but can be suppressed by one copy of picoIR. The effect of one copy of picoIR alone is shown for comparison. Mean wing area is expressed as a percentage of control (±SD).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691947&req=5

fig6: pico Interacts with ena and Modifies Actin Dynamics(A) Representative wing discs of the indicated genotypes stained for F-actin. Ectopic expression of pico or ena results in elevated F-actin, whereas ectopic picoIR reduces F-actin. pico-induced F-actin formation is dominantly suppressed by ena210. Ectopic expression, driven by en-GAL4, is limited to the posterior compartment marked by GFP. Quantitation of ratios of anterior:posterior F-actin levels is shown at the bottom of (A) (±SD).(B) Immunoblots showing levels of total actin normalized to tubulin in extracts from wing discs of the indicated genotypes. Mean actin levels from six independent experiments are expressed below as percentage of the control ± SD.(C) Ectopic expression of Myc-pico using MS1096-GAL4 results in elevated F-actin staining in the dorsal region of the wing disc where Myc-pico levels are highest. This effect is suppressed by coexpression of DN-Egfr. Quantitation of ratios of ventral:dorsal F-actin levels (±SD) is shown in the upper right corners of the upper panels.(D) Pico full-length, but not picoRA-PH, binds the Ena EVH1 domain, but not an unrelated control (NIPP1), in the yeast two-hybrid system. Interaction is indicated by blue X-gal (X) and growth on auxotrophic media at high (H) and low (L) density.(E) Ena coimmunoprecipitates with Myc-tagged pico from MS1096 > Myc-pico wing disc extracts. Lower panel shows protein immunoblot analyses of total cell lysates to control for loading.(F) Wing images of the indicated genotypes showing functional interactions between pico and ena in the wing. Wing from ena210 heterozygote resembles wild-type. ena210 dominantly suppresses the effect of ectopic pico on tissue overgrowth; compare with the effect of one copy of pico alone. Ectopic ena drives tissue overgrowth. The effect of ectopic ena is not enhanced by coexpression of pico, but can be suppressed by one copy of picoIR. The effect of one copy of picoIR alone is shown for comparison. Mean wing area is expressed as a percentage of control (±SD).

Mentions: Mammalian MRL proteins have been shown to stimulate F-actin formation without influencing total actin content (Krause et al., 2004; Lafuente et al., 2004). We found that, in the context of the wing imaginal disc, ectopic pico promoted F-actin formation, and ectopic picoIR reduced F-actin levels (Figure 6A). Total actin content was unaffected in extracts (Figure 6B), suggesting that pico regulates the ratio of G:F-actin content. EgfrDN suppressed pico-mediated changes in G:F actin ratio (Figures 6B and 6C), suggesting that this aspect of pico function is dependent on EGFR signaling.


Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

Lyulcheva E, Taylor E, Michael M, Vehlow A, Tan S, Fletcher A, Krause M, Bennett D - Dev. Cell (2008)

pico Interacts with ena and Modifies Actin Dynamics(A) Representative wing discs of the indicated genotypes stained for F-actin. Ectopic expression of pico or ena results in elevated F-actin, whereas ectopic picoIR reduces F-actin. pico-induced F-actin formation is dominantly suppressed by ena210. Ectopic expression, driven by en-GAL4, is limited to the posterior compartment marked by GFP. Quantitation of ratios of anterior:posterior F-actin levels is shown at the bottom of (A) (±SD).(B) Immunoblots showing levels of total actin normalized to tubulin in extracts from wing discs of the indicated genotypes. Mean actin levels from six independent experiments are expressed below as percentage of the control ± SD.(C) Ectopic expression of Myc-pico using MS1096-GAL4 results in elevated F-actin staining in the dorsal region of the wing disc where Myc-pico levels are highest. This effect is suppressed by coexpression of DN-Egfr. Quantitation of ratios of ventral:dorsal F-actin levels (±SD) is shown in the upper right corners of the upper panels.(D) Pico full-length, but not picoRA-PH, binds the Ena EVH1 domain, but not an unrelated control (NIPP1), in the yeast two-hybrid system. Interaction is indicated by blue X-gal (X) and growth on auxotrophic media at high (H) and low (L) density.(E) Ena coimmunoprecipitates with Myc-tagged pico from MS1096 > Myc-pico wing disc extracts. Lower panel shows protein immunoblot analyses of total cell lysates to control for loading.(F) Wing images of the indicated genotypes showing functional interactions between pico and ena in the wing. Wing from ena210 heterozygote resembles wild-type. ena210 dominantly suppresses the effect of ectopic pico on tissue overgrowth; compare with the effect of one copy of pico alone. Ectopic ena drives tissue overgrowth. The effect of ectopic ena is not enhanced by coexpression of pico, but can be suppressed by one copy of picoIR. The effect of one copy of picoIR alone is shown for comparison. Mean wing area is expressed as a percentage of control (±SD).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691947&req=5

fig6: pico Interacts with ena and Modifies Actin Dynamics(A) Representative wing discs of the indicated genotypes stained for F-actin. Ectopic expression of pico or ena results in elevated F-actin, whereas ectopic picoIR reduces F-actin. pico-induced F-actin formation is dominantly suppressed by ena210. Ectopic expression, driven by en-GAL4, is limited to the posterior compartment marked by GFP. Quantitation of ratios of anterior:posterior F-actin levels is shown at the bottom of (A) (±SD).(B) Immunoblots showing levels of total actin normalized to tubulin in extracts from wing discs of the indicated genotypes. Mean actin levels from six independent experiments are expressed below as percentage of the control ± SD.(C) Ectopic expression of Myc-pico using MS1096-GAL4 results in elevated F-actin staining in the dorsal region of the wing disc where Myc-pico levels are highest. This effect is suppressed by coexpression of DN-Egfr. Quantitation of ratios of ventral:dorsal F-actin levels (±SD) is shown in the upper right corners of the upper panels.(D) Pico full-length, but not picoRA-PH, binds the Ena EVH1 domain, but not an unrelated control (NIPP1), in the yeast two-hybrid system. Interaction is indicated by blue X-gal (X) and growth on auxotrophic media at high (H) and low (L) density.(E) Ena coimmunoprecipitates with Myc-tagged pico from MS1096 > Myc-pico wing disc extracts. Lower panel shows protein immunoblot analyses of total cell lysates to control for loading.(F) Wing images of the indicated genotypes showing functional interactions between pico and ena in the wing. Wing from ena210 heterozygote resembles wild-type. ena210 dominantly suppresses the effect of ectopic pico on tissue overgrowth; compare with the effect of one copy of pico alone. Ectopic ena drives tissue overgrowth. The effect of ectopic ena is not enhanced by coexpression of pico, but can be suppressed by one copy of picoIR. The effect of one copy of picoIR alone is shown for comparison. Mean wing area is expressed as a percentage of control (±SD).
Mentions: Mammalian MRL proteins have been shown to stimulate F-actin formation without influencing total actin content (Krause et al., 2004; Lafuente et al., 2004). We found that, in the context of the wing imaginal disc, ectopic pico promoted F-actin formation, and ectopic picoIR reduced F-actin levels (Figure 6A). Total actin content was unaffected in extracts (Figure 6B), suggesting that pico regulates the ratio of G:F-actin content. EgfrDN suppressed pico-mediated changes in G:F actin ratio (Figures 6B and 6C), suggesting that this aspect of pico function is dependent on EGFR signaling.

Bottom Line: Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner.We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal.We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK.

ABSTRACT
MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

Show MeSH
Related in: MedlinePlus