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The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development.

Nowotarski SH, McKeon N, Moser RJ, Peifer M - Mol. Biol. Cell (2014)

Bottom Line: The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells.We hypothesized that differing Enabled and/or Diaphanous activity drives these differences.Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative "fingerprint"--the protrusive profile--which our data suggest is characteristic of each actin regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

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Reducing Dia or Ena each affects AS protrusive behavior, but only Dia reduction alters the protrusive profile. (A–C) Representative movie stills, AS cells, stage 13/14 embryos expressing Moe-GFP. Scale bars, 10 μm. (A) Wild-type AS cell. Arrows, filopodia. (B) enaGC1/ena46 zygotic mutant. Arrows, short filopodia; bracket, lamellipodia. (C) dia2 zygotic mutant. Arrows, filopodia. (D) Reducing Dia increases mean filopodia number, whereas reducing Ena does not alter it. (E) Both Ena and Dia are important for mean maximum filopodium length (μm). (F) Both Ena and Dia help maintain the mean filopodial lifetime of filopodia (s). (G) Mean filopodial lifetime of the longest-lived 20% reveals that loss of Dia drastically reduces the lifetimes of the top 20%. (H) Lamellipodia area is statistically similar in all three genotypes. (I–K) Protrusive profiles. (I) In ena mutants, length and lifetime are reduced in proportion, and thus protrusive profile slope is unchanged. (J) Reducing Dia alters the protrusive profile of filopodia toward shorter lifetimes at a given length. (K) Reducing Dia in the AS yields a filopodia protrusive profile with a slope more like that of WT LE filopodia.
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Figure 7: Reducing Dia or Ena each affects AS protrusive behavior, but only Dia reduction alters the protrusive profile. (A–C) Representative movie stills, AS cells, stage 13/14 embryos expressing Moe-GFP. Scale bars, 10 μm. (A) Wild-type AS cell. Arrows, filopodia. (B) enaGC1/ena46 zygotic mutant. Arrows, short filopodia; bracket, lamellipodia. (C) dia2 zygotic mutant. Arrows, filopodia. (D) Reducing Dia increases mean filopodia number, whereas reducing Ena does not alter it. (E) Both Ena and Dia are important for mean maximum filopodium length (μm). (F) Both Ena and Dia help maintain the mean filopodial lifetime of filopodia (s). (G) Mean filopodial lifetime of the longest-lived 20% reveals that loss of Dia drastically reduces the lifetimes of the top 20%. (H) Lamellipodia area is statistically similar in all three genotypes. (I–K) Protrusive profiles. (I) In ena mutants, length and lifetime are reduced in proportion, and thus protrusive profile slope is unchanged. (J) Reducing Dia alters the protrusive profile of filopodia toward shorter lifetimes at a given length. (K) Reducing Dia in the AS yields a filopodia protrusive profile with a slope more like that of WT LE filopodia.

Mentions: Ena reduction had striking qualitative effects on filopodial behavior (Figure 7, A and B, and Supplemental Movie S8). We thus quantitated different filopodial parameters. Loss of zygotic Ena significantly reduced both filopodial maximum length and lifetime (Figure 7, E and F, and Supplemental Figure S1B), including effects on time spent extending and retracting (Supplemental Figure S1, C–F). Ena reduction had a parallel effect on the lifetime of the 20% longest-lived filopodia (Figure 7G). In contrast, lamellipodial area remained statistically indistinguishable from that of wild type (Figure 7H), and Ena reduction did not significantly alter AS filopodial number relative to that of wild type (Figure 7D), indicating that either Ena is not responsible for most filopodia initiation in the AS or residual maternal Ena is sufficient. This is in marked contrast to the LE, for which reducing Ena significantly reduces filopodia number (Homem and Peifer, 2009). Thus Ena reduction partially phenocopied FP4mito expression, suggesting that Ena does, in fact, help regulate filopodial behavior in this tissue. However, analysis of the protrusive profile of filopodia in ena mutants revealed that filopodia are both shorter and shorter-lived, and thus the slope of the protrusion profile was unchanged (Figure 7I and Supplemental Figure S2C). This suggested that Dia might elongate the remaining filopodia, consistent with the idea that Dia normally regulates the wild-type AS protrusion profile.


The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development.

Nowotarski SH, McKeon N, Moser RJ, Peifer M - Mol. Biol. Cell (2014)

Reducing Dia or Ena each affects AS protrusive behavior, but only Dia reduction alters the protrusive profile. (A–C) Representative movie stills, AS cells, stage 13/14 embryos expressing Moe-GFP. Scale bars, 10 μm. (A) Wild-type AS cell. Arrows, filopodia. (B) enaGC1/ena46 zygotic mutant. Arrows, short filopodia; bracket, lamellipodia. (C) dia2 zygotic mutant. Arrows, filopodia. (D) Reducing Dia increases mean filopodia number, whereas reducing Ena does not alter it. (E) Both Ena and Dia are important for mean maximum filopodium length (μm). (F) Both Ena and Dia help maintain the mean filopodial lifetime of filopodia (s). (G) Mean filopodial lifetime of the longest-lived 20% reveals that loss of Dia drastically reduces the lifetimes of the top 20%. (H) Lamellipodia area is statistically similar in all three genotypes. (I–K) Protrusive profiles. (I) In ena mutants, length and lifetime are reduced in proportion, and thus protrusive profile slope is unchanged. (J) Reducing Dia alters the protrusive profile of filopodia toward shorter lifetimes at a given length. (K) Reducing Dia in the AS yields a filopodia protrusive profile with a slope more like that of WT LE filopodia.
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Related In: Results  -  Collection

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Figure 7: Reducing Dia or Ena each affects AS protrusive behavior, but only Dia reduction alters the protrusive profile. (A–C) Representative movie stills, AS cells, stage 13/14 embryos expressing Moe-GFP. Scale bars, 10 μm. (A) Wild-type AS cell. Arrows, filopodia. (B) enaGC1/ena46 zygotic mutant. Arrows, short filopodia; bracket, lamellipodia. (C) dia2 zygotic mutant. Arrows, filopodia. (D) Reducing Dia increases mean filopodia number, whereas reducing Ena does not alter it. (E) Both Ena and Dia are important for mean maximum filopodium length (μm). (F) Both Ena and Dia help maintain the mean filopodial lifetime of filopodia (s). (G) Mean filopodial lifetime of the longest-lived 20% reveals that loss of Dia drastically reduces the lifetimes of the top 20%. (H) Lamellipodia area is statistically similar in all three genotypes. (I–K) Protrusive profiles. (I) In ena mutants, length and lifetime are reduced in proportion, and thus protrusive profile slope is unchanged. (J) Reducing Dia alters the protrusive profile of filopodia toward shorter lifetimes at a given length. (K) Reducing Dia in the AS yields a filopodia protrusive profile with a slope more like that of WT LE filopodia.
Mentions: Ena reduction had striking qualitative effects on filopodial behavior (Figure 7, A and B, and Supplemental Movie S8). We thus quantitated different filopodial parameters. Loss of zygotic Ena significantly reduced both filopodial maximum length and lifetime (Figure 7, E and F, and Supplemental Figure S1B), including effects on time spent extending and retracting (Supplemental Figure S1, C–F). Ena reduction had a parallel effect on the lifetime of the 20% longest-lived filopodia (Figure 7G). In contrast, lamellipodial area remained statistically indistinguishable from that of wild type (Figure 7H), and Ena reduction did not significantly alter AS filopodial number relative to that of wild type (Figure 7D), indicating that either Ena is not responsible for most filopodia initiation in the AS or residual maternal Ena is sufficient. This is in marked contrast to the LE, for which reducing Ena significantly reduces filopodia number (Homem and Peifer, 2009). Thus Ena reduction partially phenocopied FP4mito expression, suggesting that Ena does, in fact, help regulate filopodial behavior in this tissue. However, analysis of the protrusive profile of filopodia in ena mutants revealed that filopodia are both shorter and shorter-lived, and thus the slope of the protrusion profile was unchanged (Figure 7I and Supplemental Figure S2C). This suggested that Dia might elongate the remaining filopodia, consistent with the idea that Dia normally regulates the wild-type AS protrusion profile.

Bottom Line: The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells.We hypothesized that differing Enabled and/or Diaphanous activity drives these differences.Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative "fingerprint"--the protrusive profile--which our data suggest is characteristic of each actin regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

Show MeSH
Related in: MedlinePlus