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The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle.

Saunders RD, Avides MC, Howard T, Gonzalez C, Glover DM - J. Cell Biol. (1997)

Bottom Line: Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins.The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins.Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign, Cell Cycle Genetics Group, Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland.

ABSTRACT
abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

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The NH2-terminal  segment of Asp binds polymerized tubulin in vitro. (A)  SDS-PAGE of total proteins  from noninduced E. coli  strain carrying pAsp11 (lane  1), E. coli pAsp 11 induced to  express (lane 2), and E. coli  pAsp36 induced to express  (lane 3). An overlay of such a  gel is shown in B, in which  the blotted proteins have  been overlaid with polymerized tubulin that is subsequently detected with the  Bx69 monoclonal anti–β- tubulin (see Materials and  Methods). C shows an overlay assay carried out on the  indicated amounts of purified Asp segment after expression in E. coli.
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Figure 7: The NH2-terminal segment of Asp binds polymerized tubulin in vitro. (A) SDS-PAGE of total proteins from noninduced E. coli strain carrying pAsp11 (lane 1), E. coli pAsp 11 induced to express (lane 2), and E. coli pAsp36 induced to express (lane 3). An overlay of such a gel is shown in B, in which the blotted proteins have been overlaid with polymerized tubulin that is subsequently detected with the Bx69 monoclonal anti–β- tubulin (see Materials and Methods). C shows an overlay assay carried out on the indicated amounts of purified Asp segment after expression in E. coli.

Mentions: We further assessed the ability of Asp protein to bind microtubules in overlay assays in which bacterially expressed Asp fragments were separated by SDS-PAGE and blotted to a membrane that was subsequently incubated with microtubules. Bound microtubules were then detected by probing with the monoclonal anti–β-tubulin antibody Bx69. These assays were performed using either total E. coli proteins from cells expressing Asp11 and Asp36 (Fig. 7, A and B) or the corresponding purified fusion proteins (Fig. 7 C). Microtubules were observed to bind to both Asp36 and Asp11, but binding to Asp36 appears to be of a higher affinity (Fig. 7, B and C). Binding seems to be specific, as no detectable binding of microtubules to total E. coli proteins was observed in strains that had not been induced to express the fusion proteins (Fig. 7, A and B). To ensure that differences in binding did not simply reflect differences in the degree of induction of the two fusion proteins in the bacteria, we repeated the microtubule binding assay using increasing amounts of the two purified fusion proteins (Fig. 7 C). Differences in the binding affinity can be seen at all protein concentrations; Asp11 binding to microtubules was barely detectable when 1.6 μg of protein are used, whereas microtubule binding to Asp36 is readily detectable when 0.4 μg of protein were blotted to the membrane. This shows that the NH2-terminal third of the Asp protein binds more avidly to microtubules than does the central part of the molecule.


The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle.

Saunders RD, Avides MC, Howard T, Gonzalez C, Glover DM - J. Cell Biol. (1997)

The NH2-terminal  segment of Asp binds polymerized tubulin in vitro. (A)  SDS-PAGE of total proteins  from noninduced E. coli  strain carrying pAsp11 (lane  1), E. coli pAsp 11 induced to  express (lane 2), and E. coli  pAsp36 induced to express  (lane 3). An overlay of such a  gel is shown in B, in which  the blotted proteins have  been overlaid with polymerized tubulin that is subsequently detected with the  Bx69 monoclonal anti–β- tubulin (see Materials and  Methods). C shows an overlay assay carried out on the  indicated amounts of purified Asp segment after expression in E. coli.
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Related In: Results  -  Collection

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Figure 7: The NH2-terminal segment of Asp binds polymerized tubulin in vitro. (A) SDS-PAGE of total proteins from noninduced E. coli strain carrying pAsp11 (lane 1), E. coli pAsp 11 induced to express (lane 2), and E. coli pAsp36 induced to express (lane 3). An overlay of such a gel is shown in B, in which the blotted proteins have been overlaid with polymerized tubulin that is subsequently detected with the Bx69 monoclonal anti–β- tubulin (see Materials and Methods). C shows an overlay assay carried out on the indicated amounts of purified Asp segment after expression in E. coli.
Mentions: We further assessed the ability of Asp protein to bind microtubules in overlay assays in which bacterially expressed Asp fragments were separated by SDS-PAGE and blotted to a membrane that was subsequently incubated with microtubules. Bound microtubules were then detected by probing with the monoclonal anti–β-tubulin antibody Bx69. These assays were performed using either total E. coli proteins from cells expressing Asp11 and Asp36 (Fig. 7, A and B) or the corresponding purified fusion proteins (Fig. 7 C). Microtubules were observed to bind to both Asp36 and Asp11, but binding to Asp36 appears to be of a higher affinity (Fig. 7, B and C). Binding seems to be specific, as no detectable binding of microtubules to total E. coli proteins was observed in strains that had not been induced to express the fusion proteins (Fig. 7, A and B). To ensure that differences in binding did not simply reflect differences in the degree of induction of the two fusion proteins in the bacteria, we repeated the microtubule binding assay using increasing amounts of the two purified fusion proteins (Fig. 7 C). Differences in the binding affinity can be seen at all protein concentrations; Asp11 binding to microtubules was barely detectable when 1.6 μg of protein are used, whereas microtubule binding to Asp36 is readily detectable when 0.4 μg of protein were blotted to the membrane. This shows that the NH2-terminal third of the Asp protein binds more avidly to microtubules than does the central part of the molecule.

Bottom Line: Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins.The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins.Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign, Cell Cycle Genetics Group, Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland.

ABSTRACT
abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

Show MeSH
Related in: MedlinePlus