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

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.

Figure 2

Developmental pattern of expression of asp and its flanking transcripts. Northern blot analysis of asp expression (asp transcript is ∼6.5 kb). An Nco1–EcoRI fragment from the 6.5-kb asp cDNA clone was used as the probe. Poly A+ is from 0–3 h-old embryos (lane 1), 3–6 h-old embryos (lane 2), first instar larvae (lane 3), second instar larvae (lane 4), third instar larvae (lane 5), adult females (lane 6), and adult males (lane 7).

Figure 1

A cytogenetic and molecular map of the asp region. A is a representation of divisions 95 and 96 of the polytene map of chromosome arm 3R. Subdivisions A and B are indicated. The rearrangements used by Gonzalez et al. (1989) to define the asp region, In(3R)Ubx^7LLats^R and Df(3R)A117^PB197^D are indicated above the chromosomes. B shows the proximal section of the chromosome walk linked to the cytological map. B, R, and S represent BamHI, EcoRI, and SalI restriction sites, respectively. Horizontal lines below the restriction map indicate phage lambda and cosmid clones isolated from genomic libraries. Boxed fragments indicate fragments mapped by in situ hybridization. One contains the distal breakpoint of In(3R)Ubx^7LLats^R (ats), while the other is an EcoRI fragment recovered by microcloning. C is an expansion of the proximal section of the chromosome walk. The distal breakpoints of In(3R)Ubx^7LLats^R and Df(3R)Hd^γ1 are indicated. Uncertainties about the cytological limits of the deficiencies are indicated by the thinner lines. The limits of the deletion associated with the P element insertion tld allele tld^{68 –62} are indicated by the bar. D illustrates the transcription units of tld and asp as well as the two fragments used in P element mediated rescue experiments. pMBO1367 contains an 18-kb SalI fragment containing both tld and asp. pMBO1366 contains a 14-kb SalI fragment containing tld but only the 5′ portion of asp.

Figure 3

(A) The sequence of the Asp protein. Consensus sites for phosphorylation by p34^cdc2 and MAP kinases are shown. Also shown are the putative actin and calmodulin binding sites. (B) Comparison of actin binding motifs. Comparison between Asp putative actin binding site and actin binding sites present in α-actinin, spectrin, plastin, ABP120, dystrophin, and fimbrin. Only a selection of these actin binding domains is shown. Identical residues to Asp are shaded. (C) Comparison of calmodulin binding motifs. A comparison between the Asp putative calmodulin binding site and the calmodulin binding sites present in a number of other proteins. This list is not comprehensive. Identical residues have been shaded.

Figure 3

(A) The sequence of the Asp protein. Consensus sites for phosphorylation by p34^cdc2 and MAP kinases are shown. Also shown are the putative actin and calmodulin binding sites. (B) Comparison of actin binding motifs. Comparison between Asp putative actin binding site and actin binding sites present in α-actinin, spectrin, plastin, ABP120, dystrophin, and fimbrin. Only a selection of these actin binding domains is shown. Identical residues to Asp are shaded. (C) Comparison of calmodulin binding motifs. A comparison between the Asp putative calmodulin binding site and the calmodulin binding sites present in a number of other proteins. This list is not comprehensive. Identical residues have been shaded.

Figure 4

Asp expression constructs. The upper portion of the figure shows the asp cDNA indicating restriction cleavage sites and localization of the putative actin and calmodulin binding sites. Segments of the protein expressed in E. coli are indicated by shaded bars. The truncated protein expressed in flies transformed by pMBO1366 is indicated by a solid line.

Figure 5

Shortened proteins are produced by the asp¹ mutant and by pMBO1366 transformants. Western blot analysis of asp expression using the antibody Rb3133. (Lane 1) Proteins from 8 wild-type larval brains; (lane 2) proteins from 8 brains of larvae transformed by pMBO1366; (lane 3) proteins from 12 asp¹/asp¹ larval brains.

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.

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.

Figure 8

Immunolocalization of Asp in the mitotic cycle in syncytial embryos. Simultaneous staining for DNA with propidium iodide and tubulin with YL1/2 primary antibody and rhodamine-conjugated goat anti–rat IgG are shown in the first column and subsequent red channel of the merged image. Staining of Asp using Rb3133 primary antibody and FITC-conjugated goat anti–rabbit IgG is shown in the middle column and subsequent green channel in the merged image. The mitotic phases are (a) interphase, (b) prophase, (c) metaphase, (d) anaphase, and (e) telophase. The scale bar refers to the main set of panels. Single mitotic figures have been selected for the inset at a fourfold greater magnification. Bar, 25 μm.