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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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Production of modified β spectrin and DAnk2 transgenes. (A) Top, β spectrin is divided into discrete structural domains, including an N-terminal actin-binding domain (ABD), a C-terminal pleckstrin homology (PH) domain, 16 degenerate ∼106-amino acid–long spectrin repeats (ellipses), and one partial repeat near the C-terminus (Bennett and Baines, 2001). Two of the degenerate repeats (14 and 15) have been implicated in ankyrin (ank)-binding activity. Modified transgenes include α13 (ankyrin-binding repeat 15 of β spectrin replaced with repeat 12 of α spectrin; Das et al., 2006) and α8 (ankyrin-binding repeat 14 of β spectrin replaced with repeat 8 of α spectrin). Bottom, conventional ankyrins are divided into three major structural domains: an N-terminal membrane binding domain (MBD) with 24 copies of a 33-amino acid ankyrin repeat, a central spectrin-binding domain (SBD), and a C-terminal domain that diverges widely among ankyrin isoforms (Bennett and Baines, 2001). UAS-DAnk transgenes were engineered with a C-terminal EGFP tag. The DAnk2S isoform produced here contains the N-terminal ankyrin repeat domain and a truncated spectrin-binding domain. The C-terminal domain found in other Dank2 isoforms (and DAnk1) is absent in Dank2S. (B) Western blots demonstrated that the transgenes are stably expressed and are the expected size. Total protein from flies constitutively expressing β-Speca8 (lane 1, arrowhead), β-Specα13 (lane 2), or no transgene (lane 3) was probed with mouse anti-myc antibody and rat anti-α actinin as a loading control. Total protein from induced (lane 4) or uninduced (lane 5) bacteria carrying a DAnk2S-EGFP insert was probed with anti-EGFP antibody. IPTG induction strongly elevated expression of the expected DAnk2S-EGFP product (arrow). Molecular-weight standards are indicated to the left in kDa (additional 66-kDa standard indicated for different percentage gel on right). (C) Mouse anti-myc antibody staining of the β-Specα8 transgene product in the salivary gland of a mutant larva lacking endogenous β spectrin. A goat anti-mouse Texas Red–conjugated secondary antibody was used. The β-Specα8 exhibited the same polarized basolateral distribution as wild-type β spectrin (arrowhead). Scale bar, 10 μm.
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Figure 5: Production of modified β spectrin and DAnk2 transgenes. (A) Top, β spectrin is divided into discrete structural domains, including an N-terminal actin-binding domain (ABD), a C-terminal pleckstrin homology (PH) domain, 16 degenerate ∼106-amino acid–long spectrin repeats (ellipses), and one partial repeat near the C-terminus (Bennett and Baines, 2001). Two of the degenerate repeats (14 and 15) have been implicated in ankyrin (ank)-binding activity. Modified transgenes include α13 (ankyrin-binding repeat 15 of β spectrin replaced with repeat 12 of α spectrin; Das et al., 2006) and α8 (ankyrin-binding repeat 14 of β spectrin replaced with repeat 8 of α spectrin). Bottom, conventional ankyrins are divided into three major structural domains: an N-terminal membrane binding domain (MBD) with 24 copies of a 33-amino acid ankyrin repeat, a central spectrin-binding domain (SBD), and a C-terminal domain that diverges widely among ankyrin isoforms (Bennett and Baines, 2001). UAS-DAnk transgenes were engineered with a C-terminal EGFP tag. The DAnk2S isoform produced here contains the N-terminal ankyrin repeat domain and a truncated spectrin-binding domain. The C-terminal domain found in other Dank2 isoforms (and DAnk1) is absent in Dank2S. (B) Western blots demonstrated that the transgenes are stably expressed and are the expected size. Total protein from flies constitutively expressing β-Speca8 (lane 1, arrowhead), β-Specα13 (lane 2), or no transgene (lane 3) was probed with mouse anti-myc antibody and rat anti-α actinin as a loading control. Total protein from induced (lane 4) or uninduced (lane 5) bacteria carrying a DAnk2S-EGFP insert was probed with anti-EGFP antibody. IPTG induction strongly elevated expression of the expected DAnk2S-EGFP product (arrow). Molecular-weight standards are indicated to the left in kDa (additional 66-kDa standard indicated for different percentage gel on right). (C) Mouse anti-myc antibody staining of the β-Specα8 transgene product in the salivary gland of a mutant larva lacking endogenous β spectrin. A goat anti-mouse Texas Red–conjugated secondary antibody was used. The β-Specα8 exhibited the same polarized basolateral distribution as wild-type β spectrin (arrowhead). Scale bar, 10 μm.

Mentions: Previous studies established that the general ankyrin isoform in Drosophila (DAnk1) binds to the 15th repeat unit of β spectrin and that ankyrin assembles downstream of β spectrin (Das et al., 2006). A mutation affecting the ankyrin-binding activity of β spectrin repeat 15 did not affect the association of spectrin with the plasma membrane, but it shifted the distribution of DAnk1-EGFP from the plasma membrane to the cytoplasm. Here we tested the contribution of repeat 14 to ankyrin-binding activity by producing a similarly modified β spectrin transgene (Figure 5A). The coding sequence for β spectrin repeat 14 was replaced with divergent repeat 8 from α spectrin. The recombinant product was expressed from the ubiquitin promoter and examined for expression, targeting, and function in mutant rescue experiments. The myc epitope–tagged transgene product (β-Specα8) was readily detected in Western blots of total adult fly proteins (Figure 5B, lane 1) and was similar in abundance to the previously described β-Specα13 product (lane 2). Immunofluorescent staining of the transgene product in the larval salivary gland, in a mutant lacking endogenous β spectrin, revealed a bright pattern of staining of lateral contacts between cells (Figure 5C). The pattern was indistinguishable from that observed with other functional β spectrin transgene products. Thus the replacement of repeat 14 did not affect the stability or targeting of the protein in polarized epithelial cells. Nevertheless, the β-Specα8 transgene product altogether lacked biological activity in β-Specem6 rescue experiments (data not shown). Thus, unlike β-Specα13, which frequently survives to adulthood, the repeat replacement in β-Specα8 was incompatible with spectrin function in vivo.


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)

Production of modified β spectrin and DAnk2 transgenes. (A) Top, β spectrin is divided into discrete structural domains, including an N-terminal actin-binding domain (ABD), a C-terminal pleckstrin homology (PH) domain, 16 degenerate ∼106-amino acid–long spectrin repeats (ellipses), and one partial repeat near the C-terminus (Bennett and Baines, 2001). Two of the degenerate repeats (14 and 15) have been implicated in ankyrin (ank)-binding activity. Modified transgenes include α13 (ankyrin-binding repeat 15 of β spectrin replaced with repeat 12 of α spectrin; Das et al., 2006) and α8 (ankyrin-binding repeat 14 of β spectrin replaced with repeat 8 of α spectrin). Bottom, conventional ankyrins are divided into three major structural domains: an N-terminal membrane binding domain (MBD) with 24 copies of a 33-amino acid ankyrin repeat, a central spectrin-binding domain (SBD), and a C-terminal domain that diverges widely among ankyrin isoforms (Bennett and Baines, 2001). UAS-DAnk transgenes were engineered with a C-terminal EGFP tag. The DAnk2S isoform produced here contains the N-terminal ankyrin repeat domain and a truncated spectrin-binding domain. The C-terminal domain found in other Dank2 isoforms (and DAnk1) is absent in Dank2S. (B) Western blots demonstrated that the transgenes are stably expressed and are the expected size. Total protein from flies constitutively expressing β-Speca8 (lane 1, arrowhead), β-Specα13 (lane 2), or no transgene (lane 3) was probed with mouse anti-myc antibody and rat anti-α actinin as a loading control. Total protein from induced (lane 4) or uninduced (lane 5) bacteria carrying a DAnk2S-EGFP insert was probed with anti-EGFP antibody. IPTG induction strongly elevated expression of the expected DAnk2S-EGFP product (arrow). Molecular-weight standards are indicated to the left in kDa (additional 66-kDa standard indicated for different percentage gel on right). (C) Mouse anti-myc antibody staining of the β-Specα8 transgene product in the salivary gland of a mutant larva lacking endogenous β spectrin. A goat anti-mouse Texas Red–conjugated secondary antibody was used. The β-Specα8 exhibited the same polarized basolateral distribution as wild-type β spectrin (arrowhead). Scale bar, 10 μm.
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Related In: Results  -  Collection

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

Figure 5: Production of modified β spectrin and DAnk2 transgenes. (A) Top, β spectrin is divided into discrete structural domains, including an N-terminal actin-binding domain (ABD), a C-terminal pleckstrin homology (PH) domain, 16 degenerate ∼106-amino acid–long spectrin repeats (ellipses), and one partial repeat near the C-terminus (Bennett and Baines, 2001). Two of the degenerate repeats (14 and 15) have been implicated in ankyrin (ank)-binding activity. Modified transgenes include α13 (ankyrin-binding repeat 15 of β spectrin replaced with repeat 12 of α spectrin; Das et al., 2006) and α8 (ankyrin-binding repeat 14 of β spectrin replaced with repeat 8 of α spectrin). Bottom, conventional ankyrins are divided into three major structural domains: an N-terminal membrane binding domain (MBD) with 24 copies of a 33-amino acid ankyrin repeat, a central spectrin-binding domain (SBD), and a C-terminal domain that diverges widely among ankyrin isoforms (Bennett and Baines, 2001). UAS-DAnk transgenes were engineered with a C-terminal EGFP tag. The DAnk2S isoform produced here contains the N-terminal ankyrin repeat domain and a truncated spectrin-binding domain. The C-terminal domain found in other Dank2 isoforms (and DAnk1) is absent in Dank2S. (B) Western blots demonstrated that the transgenes are stably expressed and are the expected size. Total protein from flies constitutively expressing β-Speca8 (lane 1, arrowhead), β-Specα13 (lane 2), or no transgene (lane 3) was probed with mouse anti-myc antibody and rat anti-α actinin as a loading control. Total protein from induced (lane 4) or uninduced (lane 5) bacteria carrying a DAnk2S-EGFP insert was probed with anti-EGFP antibody. IPTG induction strongly elevated expression of the expected DAnk2S-EGFP product (arrow). Molecular-weight standards are indicated to the left in kDa (additional 66-kDa standard indicated for different percentage gel on right). (C) Mouse anti-myc antibody staining of the β-Specα8 transgene product in the salivary gland of a mutant larva lacking endogenous β spectrin. A goat anti-mouse Texas Red–conjugated secondary antibody was used. The β-Specα8 exhibited the same polarized basolateral distribution as wild-type β spectrin (arrowhead). Scale bar, 10 μm.
Mentions: Previous studies established that the general ankyrin isoform in Drosophila (DAnk1) binds to the 15th repeat unit of β spectrin and that ankyrin assembles downstream of β spectrin (Das et al., 2006). A mutation affecting the ankyrin-binding activity of β spectrin repeat 15 did not affect the association of spectrin with the plasma membrane, but it shifted the distribution of DAnk1-EGFP from the plasma membrane to the cytoplasm. Here we tested the contribution of repeat 14 to ankyrin-binding activity by producing a similarly modified β spectrin transgene (Figure 5A). The coding sequence for β spectrin repeat 14 was replaced with divergent repeat 8 from α spectrin. The recombinant product was expressed from the ubiquitin promoter and examined for expression, targeting, and function in mutant rescue experiments. The myc epitope–tagged transgene product (β-Specα8) was readily detected in Western blots of total adult fly proteins (Figure 5B, lane 1) and was similar in abundance to the previously described β-Specα13 product (lane 2). Immunofluorescent staining of the transgene product in the larval salivary gland, in a mutant lacking endogenous β spectrin, revealed a bright pattern of staining of lateral contacts between cells (Figure 5C). The pattern was indistinguishable from that observed with other functional β spectrin transgene products. Thus the replacement of repeat 14 did not affect the stability or targeting of the protein in polarized epithelial cells. Nevertheless, the β-Specα8 transgene product altogether lacked biological activity in β-Specem6 rescue experiments (data not shown). Thus, unlike β-Specα13, which frequently survives to adulthood, the repeat replacement in β-Specα8 was incompatible with spectrin function in vivo.

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