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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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Asp copurifies with microtubules. A shows a Western  blot fractionated by 7.5% SDS-PAGE. B shows the same protein  preparations on a Coomassie blue–stained 10% SDS-PAGE. (A  and B) Microtubule purification from 0–3-h-old Drosophila embryos after Taxol-induced polymerization. Asp was detected using the Rb3133 antibody and tubulin by the Bx69 antibody. Samples are as follows: (lane 1) 20 μg of crude embryonic protein  extract; (lane 2) 20 μg of pellet after the 16,000 g centrifugation;  (lane 3) 20 μg of protein from the supernatant fraction after sucrose gradient centrifugation; (lane 4) 10 μg of the microtubules  and associated proteins; (lane 5) 5 μg of the final microtubule  preparation; (lane 6) 10 μg of the final MAP preparation.
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Figure 6: Asp copurifies with microtubules. A shows a Western blot fractionated by 7.5% SDS-PAGE. B shows the same protein preparations on a Coomassie blue–stained 10% SDS-PAGE. (A and B) Microtubule purification from 0–3-h-old Drosophila embryos after Taxol-induced polymerization. Asp was detected using the Rb3133 antibody and tubulin by the Bx69 antibody. Samples are as follows: (lane 1) 20 μg of crude embryonic protein extract; (lane 2) 20 μg of pellet after the 16,000 g centrifugation; (lane 3) 20 μg of protein from the supernatant fraction after sucrose gradient centrifugation; (lane 4) 10 μg of the microtubules and associated proteins; (lane 5) 5 μg of the final microtubule preparation; (lane 6) 10 μg of the final MAP preparation.

Mentions: As mutations in asp affect the behavior of spindle microtubules, we sought to determine whether the Asp protein was itself microtubule associated. We purified microtubules from Drosophila embryos and took aliquots at each stage of the purification for electrophoresis and blotting onto PVDF membranes. These blots were incubated with the polyclonal serum Rb3133 and with the monoclonal antibody Bx69, which detect the Asp protein and β-tubulin, respectively (Fig. 6, A and B). Enrichment in tubulin is paralleled by an enrichment in the Asp protein. Moreover, the Asp protein seems to bind microtubules with a very high affinity as it is not released from the microtubule pellet by salt conditions that are known to dissociate most MAPs (Fig. 6, lanes 6). Asp protein is also not liberated after incubation with ATP (data not shown).


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)

Asp copurifies with microtubules. A shows a Western  blot fractionated by 7.5% SDS-PAGE. B shows the same protein  preparations on a Coomassie blue–stained 10% SDS-PAGE. (A  and B) Microtubule purification from 0–3-h-old Drosophila embryos after Taxol-induced polymerization. Asp was detected using the Rb3133 antibody and tubulin by the Bx69 antibody. Samples are as follows: (lane 1) 20 μg of crude embryonic protein  extract; (lane 2) 20 μg of pellet after the 16,000 g centrifugation;  (lane 3) 20 μg of protein from the supernatant fraction after sucrose gradient centrifugation; (lane 4) 10 μg of the microtubules  and associated proteins; (lane 5) 5 μg of the final microtubule  preparation; (lane 6) 10 μg of the final MAP preparation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139842&req=5

Figure 6: Asp copurifies with microtubules. A shows a Western blot fractionated by 7.5% SDS-PAGE. B shows the same protein preparations on a Coomassie blue–stained 10% SDS-PAGE. (A and B) Microtubule purification from 0–3-h-old Drosophila embryos after Taxol-induced polymerization. Asp was detected using the Rb3133 antibody and tubulin by the Bx69 antibody. Samples are as follows: (lane 1) 20 μg of crude embryonic protein extract; (lane 2) 20 μg of pellet after the 16,000 g centrifugation; (lane 3) 20 μg of protein from the supernatant fraction after sucrose gradient centrifugation; (lane 4) 10 μg of the microtubules and associated proteins; (lane 5) 5 μg of the final microtubule preparation; (lane 6) 10 μg of the final MAP preparation.
Mentions: As mutations in asp affect the behavior of spindle microtubules, we sought to determine whether the Asp protein was itself microtubule associated. We purified microtubules from Drosophila embryos and took aliquots at each stage of the purification for electrophoresis and blotting onto PVDF membranes. These blots were incubated with the polyclonal serum Rb3133 and with the monoclonal antibody Bx69, which detect the Asp protein and β-tubulin, respectively (Fig. 6, A and B). Enrichment in tubulin is paralleled by an enrichment in the Asp protein. Moreover, the Asp protein seems to bind microtubules with a very high affinity as it is not released from the microtubule pellet by salt conditions that are known to dissociate most MAPs (Fig. 6, lanes 6). Asp protein is also not liberated after incubation with ATP (data not shown).

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