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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.

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

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Elevating Ena or Dia activity in the AS has effects on protrusive behavior distinct from one another and from their effects at the LE. (A–C) Representative movie stills of AS cell bleach experiments in stage 14 embryos ubiquitously expressing Moe-GFP in genotypes indicated. Yellow arrows note filopodia. Scale bars, 10 μm, except B inset, 5 μm. (A) Wild-type AS cells produce filopodia (arrows) without many lamellipodia. (B) Ena overexpression increases filopodia number (arrows) but does not induce the filopodial fans seen in LE cells (B inset, arrowhead). (C) Dia∆DAD induces long filopodia (arrow, arrowheads). (D–F) Statistics via t test. (D) Mean filopodia number per micrometer of perimeter per hour. (E) Mean maximum filopodium length (μm). (F) Mean filopodial lifetime (s). (G) Mean filopodial lifetime of longest-lived 20%. (H–J) Protrusive profiles. Statistical test, ANCOVA for difference in slopes, (H) Ena overexpression in the AS alters the protrusive profile, leading to shorter-lived filopodia when controlled for length. (I) Ena overexpression in the AS yields filopodia with a protrusive profile slope resembling that of wild-type LE cells. (J) Expressing Dia∆DAD in the AS does not significantly alter the protrusive profile slope of AS cell filopodia.
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Figure 4: Elevating Ena or Dia activity in the AS has effects on protrusive behavior distinct from one another and from their effects at the LE. (A–C) Representative movie stills of AS cell bleach experiments in stage 14 embryos ubiquitously expressing Moe-GFP in genotypes indicated. Yellow arrows note filopodia. Scale bars, 10 μm, except B inset, 5 μm. (A) Wild-type AS cells produce filopodia (arrows) without many lamellipodia. (B) Ena overexpression increases filopodia number (arrows) but does not induce the filopodial fans seen in LE cells (B inset, arrowhead). (C) Dia∆DAD induces long filopodia (arrow, arrowheads). (D–F) Statistics via t test. (D) Mean filopodia number per micrometer of perimeter per hour. (E) Mean maximum filopodium length (μm). (F) Mean filopodial lifetime (s). (G) Mean filopodial lifetime of longest-lived 20%. (H–J) Protrusive profiles. Statistical test, ANCOVA for difference in slopes, (H) Ena overexpression in the AS alters the protrusive profile, leading to shorter-lived filopodia when controlled for length. (I) Ena overexpression in the AS yields filopodia with a protrusive profile slope resembling that of wild-type LE cells. (J) Expressing Dia∆DAD in the AS does not significantly alter the protrusive profile slope of AS cell filopodia.

Mentions: The foregoing data combined with our earlier work suggest that Ena may be the dominant actin elongator acting at the LE. The differences in protrusive profile between the wild-type LE and AS, along with the shift in protrusive profile induced by Dia at the LE, led us to hypothesize that Dia may be more important in the AS. To begin to test this, we elevated either Ena or Dia activity in AS cells, expressing RFP-Ena or HA-Dia∆DAD specifically in the AS using c381-GAL4, and driving sqh-Moe-GFP to visualize protrusions and compare them to wild type (Figure 4, A–C, and Supplemental Movie S6). Elevating levels of these actin regulators did not prevent completion of dorsal closure. HA-Dia∆DAD did not substantially change either the overall process of dorsal closure or its timing, as assessed by area change or canthi migration within the last 90 min of closure (Supplemental Figure S4, A and C–E). Overexpressing Ena did not alter overall closure or the change in AS area, but it did increase the rate of closure as determined by canthi distance (Supplemental Figure S4, A, B, D, and E, and Supplemental Movie S7).


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)

Elevating Ena or Dia activity in the AS has effects on protrusive behavior distinct from one another and from their effects at the LE. (A–C) Representative movie stills of AS cell bleach experiments in stage 14 embryos ubiquitously expressing Moe-GFP in genotypes indicated. Yellow arrows note filopodia. Scale bars, 10 μm, except B inset, 5 μm. (A) Wild-type AS cells produce filopodia (arrows) without many lamellipodia. (B) Ena overexpression increases filopodia number (arrows) but does not induce the filopodial fans seen in LE cells (B inset, arrowhead). (C) Dia∆DAD induces long filopodia (arrow, arrowheads). (D–F) Statistics via t test. (D) Mean filopodia number per micrometer of perimeter per hour. (E) Mean maximum filopodium length (μm). (F) Mean filopodial lifetime (s). (G) Mean filopodial lifetime of longest-lived 20%. (H–J) Protrusive profiles. Statistical test, ANCOVA for difference in slopes, (H) Ena overexpression in the AS alters the protrusive profile, leading to shorter-lived filopodia when controlled for length. (I) Ena overexpression in the AS yields filopodia with a protrusive profile slope resembling that of wild-type LE cells. (J) Expressing Dia∆DAD in the AS does not significantly alter the protrusive profile slope of AS cell filopodia.
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Related In: Results  -  Collection

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Figure 4: Elevating Ena or Dia activity in the AS has effects on protrusive behavior distinct from one another and from their effects at the LE. (A–C) Representative movie stills of AS cell bleach experiments in stage 14 embryos ubiquitously expressing Moe-GFP in genotypes indicated. Yellow arrows note filopodia. Scale bars, 10 μm, except B inset, 5 μm. (A) Wild-type AS cells produce filopodia (arrows) without many lamellipodia. (B) Ena overexpression increases filopodia number (arrows) but does not induce the filopodial fans seen in LE cells (B inset, arrowhead). (C) Dia∆DAD induces long filopodia (arrow, arrowheads). (D–F) Statistics via t test. (D) Mean filopodia number per micrometer of perimeter per hour. (E) Mean maximum filopodium length (μm). (F) Mean filopodial lifetime (s). (G) Mean filopodial lifetime of longest-lived 20%. (H–J) Protrusive profiles. Statistical test, ANCOVA for difference in slopes, (H) Ena overexpression in the AS alters the protrusive profile, leading to shorter-lived filopodia when controlled for length. (I) Ena overexpression in the AS yields filopodia with a protrusive profile slope resembling that of wild-type LE cells. (J) Expressing Dia∆DAD in the AS does not significantly alter the protrusive profile slope of AS cell filopodia.
Mentions: The foregoing data combined with our earlier work suggest that Ena may be the dominant actin elongator acting at the LE. The differences in protrusive profile between the wild-type LE and AS, along with the shift in protrusive profile induced by Dia at the LE, led us to hypothesize that Dia may be more important in the AS. To begin to test this, we elevated either Ena or Dia activity in AS cells, expressing RFP-Ena or HA-Dia∆DAD specifically in the AS using c381-GAL4, and driving sqh-Moe-GFP to visualize protrusions and compare them to wild type (Figure 4, A–C, and Supplemental Movie S6). Elevating levels of these actin regulators did not prevent completion of dorsal closure. HA-Dia∆DAD did not substantially change either the overall process of dorsal closure or its timing, as assessed by area change or canthi migration within the last 90 min of closure (Supplemental Figure S4, A and C–E). Overexpressing Ena did not alter overall closure or the change in AS area, but it did increase the rate of closure as determined by canthi distance (Supplemental Figure S4, A, B, D, and E, and Supplemental Movie S7).

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