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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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Neuronal expression of UAS-β-Spec95 was sufficient to rescue the lethality of β spectrin mutants. (A) Rescue cross. The neuronal driver elav-Gal4 was recombined onto the X chromosome of a female fly heterozygous for the spectrin loss-of-function mutation β-Specem6. Heterozygous elav-Gal4-β-Specem6/FM7; +/+ female flies were then crossed to male flies carrying the myc-tagged wild-type transgene (+/Y; UAS-β-Spec95/ UAS-β-Spec95). Successful rescue was scored by the presence of a non-FM7 F1 male class (which lacks endogenous β spectrin). (B) Adult rescue flies. Flies rescued with the UAS-β-Spec95 transgene at 25°C were generally smaller than wild-type siblings (FM7/Y) and had wing unfolding defects similar to wild-type flies that overexpressed UAS-β-Spec95 (Table 1). In contrast, flies rescued with UAS-β-Spec493 at any temperature or with UAS-β-Spec95 at 22°C did not exhibit wing unfolding or body size defects. (C) Quantitative analysis of rescue. Neuronal expression of either the UAS-β-Spec95 or the UAS-β-Spec493 transgenes (both at 25°C) efficiently rescued the lethal β-Specem6 mutation at about the same rate as a ubiquitously expressed β spectrin transgene described previously (Dubreuil et al., 2000). Expression of UAS-α-Spec27 (negative control) did not produce the rescue class of male progeny.
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Figure 3: Neuronal expression of UAS-β-Spec95 was sufficient to rescue the lethality of β spectrin mutants. (A) Rescue cross. The neuronal driver elav-Gal4 was recombined onto the X chromosome of a female fly heterozygous for the spectrin loss-of-function mutation β-Specem6. Heterozygous elav-Gal4-β-Specem6/FM7; +/+ female flies were then crossed to male flies carrying the myc-tagged wild-type transgene (+/Y; UAS-β-Spec95/ UAS-β-Spec95). Successful rescue was scored by the presence of a non-FM7 F1 male class (which lacks endogenous β spectrin). (B) Adult rescue flies. Flies rescued with the UAS-β-Spec95 transgene at 25°C were generally smaller than wild-type siblings (FM7/Y) and had wing unfolding defects similar to wild-type flies that overexpressed UAS-β-Spec95 (Table 1). In contrast, flies rescued with UAS-β-Spec493 at any temperature or with UAS-β-Spec95 at 22°C did not exhibit wing unfolding or body size defects. (C) Quantitative analysis of rescue. Neuronal expression of either the UAS-β-Spec95 or the UAS-β-Spec493 transgenes (both at 25°C) efficiently rescued the lethal β-Specem6 mutation at about the same rate as a ubiquitously expressed β spectrin transgene described previously (Dubreuil et al., 2000). Expression of UAS-α-Spec27 (negative control) did not produce the rescue class of male progeny.

Mentions: The failure to phenocopy lethality by dsRNA knockdown of β spectrin in nonneuronal cells together with the recent demonstration that neuronal ankyrin was required for viability in Drosophila (Koch et al., 2008; Pielage et al., 2008) led us to revisit the question of whether or not the essential function of β spectrin resides in the nervous system. In previous mutant rescue experiments we scored the ability of a ubiquitously expressed autosomal β spectrin transgene to rescue F1 males carrying the lethal β spectrin allele on X (Das et al., 2006). The same single-generation rescue strategy was used here to rescue males carrying the lethal β spectrin allele by expressing UAS-β-Spec95 with the neuronal driver elav-Gal4. The elav-Gal4 insertion is also X-linked; therefore, we produced a recombinant chromosome carrying both the lethal β-Specem6 mutation and elav-Gal4 (Figure 3A). Heterozygous females carrying the recombinant mutant chromosome over a wild-type balancer chromosome (FM7) were crossed to homozygous UAS-β-Spec95 males. If neuronal expression of UAS-β-Spec95 was not sufficient to rescue lethality, then all of the surviving F1 male progeny should have carried the balancer chromosome with its wild-type copy of the β spectrin gene. However, nervous system expression of UAS-β-Spec95 was sufficient to allow a high rate of survival of F1 males carrying the β-Specem6-elav-Gal4 chromosome (Figure 3, B and C). In initial experiments where flies were reared at 25°C the rescued males appeared somewhat unhealthy (crumpled wings, rough eyes, small size; Figure 3B) and were short-lived. Nevertheless, these males were fertile and thus represent a significant rescue of the embryonic lethality normally associated with the β spectrin loss-of-function mutation. The nervous system–specific expression of elav in the embryo has been rigorously characterized (Berger et al., 2007). Control experiments using the reporter UAS-mCD8-GFP verified that expression of elav-Gal4 was limited to the nervous system during larval development, except for promiscuous expression in the salivary gland (Figure S3).


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)

Neuronal expression of UAS-β-Spec95 was sufficient to rescue the lethality of β spectrin mutants. (A) Rescue cross. The neuronal driver elav-Gal4 was recombined onto the X chromosome of a female fly heterozygous for the spectrin loss-of-function mutation β-Specem6. Heterozygous elav-Gal4-β-Specem6/FM7; +/+ female flies were then crossed to male flies carrying the myc-tagged wild-type transgene (+/Y; UAS-β-Spec95/ UAS-β-Spec95). Successful rescue was scored by the presence of a non-FM7 F1 male class (which lacks endogenous β spectrin). (B) Adult rescue flies. Flies rescued with the UAS-β-Spec95 transgene at 25°C were generally smaller than wild-type siblings (FM7/Y) and had wing unfolding defects similar to wild-type flies that overexpressed UAS-β-Spec95 (Table 1). In contrast, flies rescued with UAS-β-Spec493 at any temperature or with UAS-β-Spec95 at 22°C did not exhibit wing unfolding or body size defects. (C) Quantitative analysis of rescue. Neuronal expression of either the UAS-β-Spec95 or the UAS-β-Spec493 transgenes (both at 25°C) efficiently rescued the lethal β-Specem6 mutation at about the same rate as a ubiquitously expressed β spectrin transgene described previously (Dubreuil et al., 2000). Expression of UAS-α-Spec27 (negative control) did not produce the rescue class of male progeny.
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Related In: Results  -  Collection

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Figure 3: Neuronal expression of UAS-β-Spec95 was sufficient to rescue the lethality of β spectrin mutants. (A) Rescue cross. The neuronal driver elav-Gal4 was recombined onto the X chromosome of a female fly heterozygous for the spectrin loss-of-function mutation β-Specem6. Heterozygous elav-Gal4-β-Specem6/FM7; +/+ female flies were then crossed to male flies carrying the myc-tagged wild-type transgene (+/Y; UAS-β-Spec95/ UAS-β-Spec95). Successful rescue was scored by the presence of a non-FM7 F1 male class (which lacks endogenous β spectrin). (B) Adult rescue flies. Flies rescued with the UAS-β-Spec95 transgene at 25°C were generally smaller than wild-type siblings (FM7/Y) and had wing unfolding defects similar to wild-type flies that overexpressed UAS-β-Spec95 (Table 1). In contrast, flies rescued with UAS-β-Spec493 at any temperature or with UAS-β-Spec95 at 22°C did not exhibit wing unfolding or body size defects. (C) Quantitative analysis of rescue. Neuronal expression of either the UAS-β-Spec95 or the UAS-β-Spec493 transgenes (both at 25°C) efficiently rescued the lethal β-Specem6 mutation at about the same rate as a ubiquitously expressed β spectrin transgene described previously (Dubreuil et al., 2000). Expression of UAS-α-Spec27 (negative control) did not produce the rescue class of male progeny.
Mentions: The failure to phenocopy lethality by dsRNA knockdown of β spectrin in nonneuronal cells together with the recent demonstration that neuronal ankyrin was required for viability in Drosophila (Koch et al., 2008; Pielage et al., 2008) led us to revisit the question of whether or not the essential function of β spectrin resides in the nervous system. In previous mutant rescue experiments we scored the ability of a ubiquitously expressed autosomal β spectrin transgene to rescue F1 males carrying the lethal β spectrin allele on X (Das et al., 2006). The same single-generation rescue strategy was used here to rescue males carrying the lethal β spectrin allele by expressing UAS-β-Spec95 with the neuronal driver elav-Gal4. The elav-Gal4 insertion is also X-linked; therefore, we produced a recombinant chromosome carrying both the lethal β-Specem6 mutation and elav-Gal4 (Figure 3A). Heterozygous females carrying the recombinant mutant chromosome over a wild-type balancer chromosome (FM7) were crossed to homozygous UAS-β-Spec95 males. If neuronal expression of UAS-β-Spec95 was not sufficient to rescue lethality, then all of the surviving F1 male progeny should have carried the balancer chromosome with its wild-type copy of the β spectrin gene. However, nervous system expression of UAS-β-Spec95 was sufficient to allow a high rate of survival of F1 males carrying the β-Specem6-elav-Gal4 chromosome (Figure 3, B and C). In initial experiments where flies were reared at 25°C the rescued males appeared somewhat unhealthy (crumpled wings, rough eyes, small size; Figure 3B) and were short-lived. Nevertheless, these males were fertile and thus represent a significant rescue of the embryonic lethality normally associated with the β spectrin loss-of-function mutation. The nervous system–specific expression of elav in the embryo has been rigorously characterized (Berger et al., 2007). Control experiments using the reporter UAS-mCD8-GFP verified that expression of elav-Gal4 was limited to the nervous system during larval development, except for promiscuous expression in the salivary gland (Figure S3).

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