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Transgene rescue identifies an essential function for Drosophila beta spectrin in the nervous system and a selective requirement for ankyrin-2-binding activity.

Mazock GH, Das A, Base C, Dubreuil RR - Mol. Biol. Cell (2010)

Bottom Line: The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation.Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability.Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential beta spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a beta spectrin transgene or to knock down beta spectrin expression with dsRNA. The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation. Thus beta spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.

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Overexpression of β spectrin in a wild-type background produced dominant negative effects in vivo. (A) Overexpression of UAS-α and UAS-β spectrin transgenes in the wing disk. Top, overexpression of the UAS-α-Spec37 transgene in the wing disk driven by MS1096-Gal4 did not affect wing development. Bottom, overexpression of the UAS-β-Spec95 transgene blocked normal wing development. (B) Overexpression of the UAS-β-Spec95 transgene driven by the neuronal elav-Gal4 caused defects in wing inflation/unfolding. (C) Overexpression of UAS-β-Spec95 in the salivary gland. Left, in control salivary glands, β spectrin was localized to the basolateral membranes of epithelial cells. β spectrin was detected with a rabbit anti-β spectrin antibody and a Cy3-labeled secondary antibody. Right, UAS-β-Spec95 was expressed in the salivary gland (sg) via basal activity of heat-shock Gal4, which resulted in perturbed epithelial cell polarity. β spectrin was aberrantly associated with the apical membrane domain, and some cells no longer showed cuboidal morphology. β-Spec95 was detected with mouse anti-myc antibody and Cy3-labeled secondary antibody. Scale bar, 10 μm.
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Figure 1: Overexpression of β spectrin in a wild-type background produced dominant negative effects in vivo. (A) Overexpression of UAS-α and UAS-β spectrin transgenes in the wing disk. Top, overexpression of the UAS-α-Spec37 transgene in the wing disk driven by MS1096-Gal4 did not affect wing development. Bottom, overexpression of the UAS-β-Spec95 transgene blocked normal wing development. (B) Overexpression of the UAS-β-Spec95 transgene driven by the neuronal elav-Gal4 caused defects in wing inflation/unfolding. (C) Overexpression of UAS-β-Spec95 in the salivary gland. Left, in control salivary glands, β spectrin was localized to the basolateral membranes of epithelial cells. β spectrin was detected with a rabbit anti-β spectrin antibody and a Cy3-labeled secondary antibody. Right, UAS-β-Spec95 was expressed in the salivary gland (sg) via basal activity of heat-shock Gal4, which resulted in perturbed epithelial cell polarity. β spectrin was aberrantly associated with the apical membrane domain, and some cells no longer showed cuboidal morphology. β-Spec95 was detected with mouse anti-myc antibody and Cy3-labeled secondary antibody. Scale bar, 10 μm.

Mentions: The experiments reported here grew from the initial observation that overexpression of β spectrin was disruptive to wing development. When a UAS-β spectrin transgene (UAS-β-Spec95) was overexpressed in the developing wing disk using MS1096-Gal4 there was a gross failure in wing formation (Figure 1A, bottom). In contrast to the effects of β spectrin overexpression, α spectrin overexpression with the same driver (Figure S1A) had no apparent effect on wing development (Figure 1A, top). Thus the disruptive effects were not a trivial consequence of overexpressing a large protein product.


Transgene rescue identifies an essential function for Drosophila beta spectrin in the nervous system and a selective requirement for ankyrin-2-binding activity.

Mazock GH, Das A, Base C, Dubreuil RR - Mol. Biol. Cell (2010)

Overexpression of β spectrin in a wild-type background produced dominant negative effects in vivo. (A) Overexpression of UAS-α and UAS-β spectrin transgenes in the wing disk. Top, overexpression of the UAS-α-Spec37 transgene in the wing disk driven by MS1096-Gal4 did not affect wing development. Bottom, overexpression of the UAS-β-Spec95 transgene blocked normal wing development. (B) Overexpression of the UAS-β-Spec95 transgene driven by the neuronal elav-Gal4 caused defects in wing inflation/unfolding. (C) Overexpression of UAS-β-Spec95 in the salivary gland. Left, in control salivary glands, β spectrin was localized to the basolateral membranes of epithelial cells. β spectrin was detected with a rabbit anti-β spectrin antibody and a Cy3-labeled secondary antibody. Right, UAS-β-Spec95 was expressed in the salivary gland (sg) via basal activity of heat-shock Gal4, which resulted in perturbed epithelial cell polarity. β spectrin was aberrantly associated with the apical membrane domain, and some cells no longer showed cuboidal morphology. β-Spec95 was detected with mouse anti-myc antibody and Cy3-labeled secondary antibody. Scale bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921109&req=5

Figure 1: Overexpression of β spectrin in a wild-type background produced dominant negative effects in vivo. (A) Overexpression of UAS-α and UAS-β spectrin transgenes in the wing disk. Top, overexpression of the UAS-α-Spec37 transgene in the wing disk driven by MS1096-Gal4 did not affect wing development. Bottom, overexpression of the UAS-β-Spec95 transgene blocked normal wing development. (B) Overexpression of the UAS-β-Spec95 transgene driven by the neuronal elav-Gal4 caused defects in wing inflation/unfolding. (C) Overexpression of UAS-β-Spec95 in the salivary gland. Left, in control salivary glands, β spectrin was localized to the basolateral membranes of epithelial cells. β spectrin was detected with a rabbit anti-β spectrin antibody and a Cy3-labeled secondary antibody. Right, UAS-β-Spec95 was expressed in the salivary gland (sg) via basal activity of heat-shock Gal4, which resulted in perturbed epithelial cell polarity. β spectrin was aberrantly associated with the apical membrane domain, and some cells no longer showed cuboidal morphology. β-Spec95 was detected with mouse anti-myc antibody and Cy3-labeled secondary antibody. Scale bar, 10 μm.
Mentions: The experiments reported here grew from the initial observation that overexpression of β spectrin was disruptive to wing development. When a UAS-β spectrin transgene (UAS-β-Spec95) was overexpressed in the developing wing disk using MS1096-Gal4 there was a gross failure in wing formation (Figure 1A, bottom). In contrast to the effects of β spectrin overexpression, α spectrin overexpression with the same driver (Figure S1A) had no apparent effect on wing development (Figure 1A, top). Thus the disruptive effects were not a trivial consequence of overexpressing a large protein product.

Bottom Line: The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation.Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability.Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential beta spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a beta spectrin transgene or to knock down beta spectrin expression with dsRNA. The results show that 1) overexpression of beta spectrin in most of the cell types studied was lethal; 2) knockdown of beta spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-beta spectrin transgene was sufficient to overcome the lethality of a loss-of-function beta spectrin mutation. Thus beta spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for beta spectrin function in vivo.

Show MeSH
Related in: MedlinePlus