Limits...
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.

Show MeSH

Related in: MedlinePlus

Lifespan of UAS-β-Spec493-rescued β-Specem6 mutants. Age at death was plotted for β-Specem6 mutants rescued by the β-Spec493 transgene (blue) compared with FM7 controls (red) all reared at 25°C. Similar results were obtained with the β-Spec95 transgene (not shown). Adult progeny from a representative rescue cross were scored daily to determine posteclosion age at time of death. Neuron-specific expression of β-Spec493 was sufficient to allow development of β spectrin mutants to adulthood, but the rescued flies were unusually short-lived.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2921109&req=5

Figure 4: Lifespan of UAS-β-Spec493-rescued β-Specem6 mutants. Age at death was plotted for β-Specem6 mutants rescued by the β-Spec493 transgene (blue) compared with FM7 controls (red) all reared at 25°C. Similar results were obtained with the β-Spec95 transgene (not shown). Adult progeny from a representative rescue cross were scored daily to determine posteclosion age at time of death. Neuron-specific expression of β-Spec493 was sufficient to allow development of β spectrin mutants to adulthood, but the rescued flies were unusually short-lived.

Mentions: One possible explanation for the apparent poor health of the mutants rescued by expression of β spectrin in the nervous system was that β-Spec95 expression was too limited; however, unexpectedly, we found that the opposite was true. The appearance of the rescued mutants improved when the level of β spectrin transgene expression was reduced. First, we repeated the rescue experiments using an independently produced UAS-β spectrin transgene (UAS-β-Spec493; Hulsmeier et al., 2007) that failed to produce the dominant negative phenotypes observed with UAS-β-Spec95, because it is expressed at a lower level (data not shown). When rescue experiments were carried out with elav-Gal4–driven UAS-β-Spec493 expression the rescue flies appeared as healthy as controls, having properly unfolded wings and a full-size thorax and abdomen (Figure 3B). Thus the defects observed in initial experiments were actually due to β spectrin overexpression, rather than insufficient expression. On the basis of these observations, we reexamined the rescue activity of UAS-β-Spec95 expression by carrying out the rescue cross at reduced temperature (22°C). The level of transgene expression was reduced (Figure S1B), and we found that once again the appearance of the rescued flies was normal (Figure 3B). The flies were fertile, and it has been possible to propagate them as a stable line for many generations. Nevertheless, although the rescue flies appeared healthier when transgene expression level was lowered, their lifespan was still reduced to about the same extent as with UAS-β-Spec95 rescue at 25°C (Figure 4). This effect was not a trivial consequence of β spectrin overexpression, because reduction of lifespan was not observed when either UAS transgene was expressed in a wild-type background (not shown).


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)

Lifespan of UAS-β-Spec493-rescued β-Specem6 mutants. Age at death was plotted for β-Specem6 mutants rescued by the β-Spec493 transgene (blue) compared with FM7 controls (red) all reared at 25°C. Similar results were obtained with the β-Spec95 transgene (not shown). Adult progeny from a representative rescue cross were scored daily to determine posteclosion age at time of death. Neuron-specific expression of β-Spec493 was sufficient to allow development of β spectrin mutants to adulthood, but the rescued flies were unusually short-lived.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921109&req=5

Figure 4: Lifespan of UAS-β-Spec493-rescued β-Specem6 mutants. Age at death was plotted for β-Specem6 mutants rescued by the β-Spec493 transgene (blue) compared with FM7 controls (red) all reared at 25°C. Similar results were obtained with the β-Spec95 transgene (not shown). Adult progeny from a representative rescue cross were scored daily to determine posteclosion age at time of death. Neuron-specific expression of β-Spec493 was sufficient to allow development of β spectrin mutants to adulthood, but the rescued flies were unusually short-lived.
Mentions: One possible explanation for the apparent poor health of the mutants rescued by expression of β spectrin in the nervous system was that β-Spec95 expression was too limited; however, unexpectedly, we found that the opposite was true. The appearance of the rescued mutants improved when the level of β spectrin transgene expression was reduced. First, we repeated the rescue experiments using an independently produced UAS-β spectrin transgene (UAS-β-Spec493; Hulsmeier et al., 2007) that failed to produce the dominant negative phenotypes observed with UAS-β-Spec95, because it is expressed at a lower level (data not shown). When rescue experiments were carried out with elav-Gal4–driven UAS-β-Spec493 expression the rescue flies appeared as healthy as controls, having properly unfolded wings and a full-size thorax and abdomen (Figure 3B). Thus the defects observed in initial experiments were actually due to β spectrin overexpression, rather than insufficient expression. On the basis of these observations, we reexamined the rescue activity of UAS-β-Spec95 expression by carrying out the rescue cross at reduced temperature (22°C). The level of transgene expression was reduced (Figure S1B), and we found that once again the appearance of the rescued flies was normal (Figure 3B). The flies were fertile, and it has been possible to propagate them as a stable line for many generations. Nevertheless, although the rescue flies appeared healthier when transgene expression level was lowered, their lifespan was still reduced to about the same extent as with UAS-β-Spec95 rescue at 25°C (Figure 4). This effect was not a trivial consequence of β spectrin overexpression, because reduction of lifespan was not observed when either UAS transgene was expressed in a wild-type background (not shown).

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