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© The Rockefeller University Press,
0021-9525/1997//881 $5.00
The Journal of Cell Biology, Volume 137, Number 4,
, 1997 881-890
Article |
The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle
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
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 p34cdc2 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.
Mutations in abnormal spindle (asp)1 lead to defects in mitosis at a variety of developmental stages as well as in male meiosis. The late larval–pupal lethality seen with strong hypomorphic alleles of asp is typical of mutations in Drosophila genes encoding stable proteins essential for mitosis. Homozygous individuals receive sufficient maternal gene product from their heterozygous mothers to permit normal embryonic development to proceed. However, there is insufficient functional gene product to permit mitosis during larval and pupal stages, when the imaginal discs and abdominal histoblast nests proliferate to form adult structures. Rare escapers may display a number of cuticular abnormalities associated with cell death, such as missing bristles, misshapen tergites and sternites, and roughened eyes. The maternal contribution of Asp protein has been demonstrated directly in cuticular clones in which there has been the loss of a marked Y chromosome as a result of nondisjunction due to the mutation (Carmena et al., 1991). The frequency and size of such clones correlate inversely with the amount of maternally contributed asp+ gene product.
The asp locus was originally identified through a late larval lethal mutation that causes defects in both mitosis and meiosis (Ripoll et al., 1985). Several mitotic abnormalities may be observed in third instar larval neuroblasts from asp homozygotes. There is an increased mitotic index and a high frequency of polyploid cells. Many metaphase figures have very highly condensed chromosomes, suggestive of a delay in progress through metaphase, and anaphase figures appear abnormally broad. asp1 animals show reduced fertility, and a high frequency of nondisjunction has been observed in both divisions of male meiosis. Phase contrast microscopic analysis of meiosis in homozygous asp males revealed the abnormalities of spindle structure that gave the locus its name (Ripoll et al., 1985) and that were subsequently confirmed by electron (Casal et al., 1990) and immunofluorescent microscopy (Gonzalez et al., 1990). Similar spindle abnormalities were seen in mitosis in the larval neuroblasts of strong asp hypomorphs (Gonzalez et al., 1990). Typically these mitotic cells have long wavy arrays of microtubules. Hemi-spindles are frequently observed in which a long dense array of microtubules is nucleated from a single centrosome.
Animals transheterozygous for particular mutant alleles of asp show an increased frequency of survival to adulthood (Gonzalez et al., 1990). However, survivors are female sterile and produce syncytial embryos displaying a number of abnormalities in nuclear divisions. One class of these embryos has no DNA, as judged by fluorescent staining, presumably reflecting failure of germline mitosis. A second class shows a variety of problems in nuclear division, including an abnormal ratio of centrosomes to nuclei and broadened or fused spindles with wavy microtubules.
Taken together, the various phenotypes seen in asp homozygotes suggest that the asp gene product may be involved in some aspect of spindle microtubule dynamics. In this paper we show that asp encodes a microtubule-associated protein that localizes to the polar regions of the spindle early in mitosis and the midbody at telophase.
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Materials and Methods |
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Chromosome Walking
Genomic clones were isolated from lambda libraries in Lambda dash (Stratagene, La Jolla, CA), and cosmid libraries were those constructed using the vectors Smart2 (Speek et al., 1988) or Lorist6 (Siden-Kiamos et al., 1990). The initial screening was carried out with cloned DNA arising from the first microcloning experiment. Subsequent steps of the walk used terminal RNA probes synthesized from the promoters at the ends of the cloning vectors. Periodically, the progress of the walk was monitored by in situ hybridization as described (Saunders et al., 1989). In situ hybridization of fragments from the chromosome walk to In(3R+3L)ats chromosomes (with which the deficiency-bearing In(3R)Ubx7LLatsR was synthesized; Gonzalez et al., 1989) enabled the location of the distal breakpoint of the deficiency In(3R)Ubx7LLatsR to be determined. To map the distal breakpoint of Df(3R)Hd
1, in situ hybridization with fluorescent labels (Saunders, 1994) was used to visualize signals derived from one or both homologues of 3R.
cDNAs were subsequently isolated by using segments of the walk to screen a testis cDNA library, kindly provided by T. Hazelrigg that had been constructed by a combination of random and oligo-dT priming in the vector 
ZAP and also embryonic cDNA libraries (provided by Brown, N.; Brown and Kafatos, 1988).
P element–mediated Rescue of asp1 Mutants
Rescue experiments were carried out using two Drosophila melanogaster lines containing homozygous second chromosome insertions of the P element transformation vector plasmids pMBO1366 and pMBO1367, respectively (Shimell et al., 1991). Plasmid pMBO1367 contains an 18-kb SalI fragment that includes both the tld and asp transcripts; pMBO1366 contains a 14-kb SalI fragment that includes the tld transcript but only the 5' half of the asp transcript. Females with the genotype +/+; asp1 red/TM6B were crossed to 1367/1367; +/+ males. 1367/+; asp1 red/+ males were selected from the F1 progeny and crossed to +/ +; asp1 red/TM6B females to obtain asp1 red/asp1 red individuals. 1367; asp1/asp1 animals did not show any morphological defect and were fully fertile, indicating that the 1367 insertion rescues the asp1 homozygous lethal phenotype. As a control, the same crosses were carried out using 1366 transformants. This insertion failed to rescue the asp1 mutation.
Sequence Determination and Analysis
Sequencing was carried out using the dideoxynucleotide chain termination procedure (Sambrook et al., 1989). Template clones for sequencing were produced by exonuclease III deletion (Sambrook et al., 1989) of inserts subcloned in Bluescript SK+ and KS+. Sequences were assembled using Microgenie (Beckmann Instruments, Inc., Fullerton, CA) and Lasergene (DNASTAR Inc., Madison, WI) software. Searches of GenBank/ EMBL/DDBJ database were conducted using BLAST with the MRC human genome mapping project computer. The protein sequence was analyzed using DOMAIN, SAPS, and COILS programs.
Expression of Asp Protein in Escherichia coli and Raising Polyclonal Antibodies
Plasmid pASP36 was constructed by inserting the NcoI–BamHI fragment from p6a (see Fig. 2) into the expression vector pET23d (Invitrogen Corp., San Diego, CA). Conditions for culture and induction were as described by the supplier. The polypeptide expressed by this clone represents the NH2-terminal portion of the Asp protein and does not include the putative actin binding domain. This polypeptide was insoluble and was purified by preparing inclusion bodies, separating the proteins by polyacrylamide gel electrophoresis, excising the appropriate band from the gel, and electroeluting the protein as described by Leppard et al. (1983). Polyclonal sera were prepared by injecting rabbits as described (Harlow and Lane, 1988).
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Embryo Staining
Embryos were fixed and stained as described by Gonzalez and Glover (1993). Propidium iodide was used to visualize DNA; the rat monoclonal antibody YL1/2 (Kilmartin et al., 1982) was used to stain microtubules, and the rabbit polyclonal antibody Rb3133 was used to stain the Asp protein. Staining was revealed using Texas red–conjugated goat anti–rat antibody and fluorescein-conjugated goat anti–rabbit antibody. All commercial antibodies were obtained from Jackson ImmunoResearch Labs, Inc. (West Grove, PA). Images were collected using an Optiphot microscope (Nikon Inc., Garden City, NY) with a confocal scanning head (model 600; BioRad, Richmond, CA) and merged using Adobe Photoshop V2.5.
Microtubule Preparation
Microtubules were purified from 0–3-h-old Drosophila embryos essentially as described by Goldstein et al. (1986). Embryos were collected, dechorionated in 50% bleach, and washed with tap water and then with lysis buffer. About 3 ml of embryos were homogenized in 2 vol of ice cold lysis buffer (0.1 M Pipes/NaOH, pH 6.6, 5 mM EGTA, 1 mM MgSO4, 0.9 M glycerol, 1 mM DTT, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin) with a Dounce homogenizer. The microtubules were depolymerized by incubation on ice for 15 min, and the extract was then centrifuged at 16,000 g for 30 min at 4°C. The supernatant was recentrifuged at 135,000 g for 90 min at 4°C. Microtubules in this latter supernatant were polymerized by addition of GTP, to a final concentration of 1 mM, addition of taxol, to a final concentration of 20 mM, and incubation at room temperature for 30 min. 3-ml aliquots of extract was layered on top of 3-ml 15% sucrose cushions prepared in lysis buffer supplemented with 20 mM taxol and 1 mM GTP. After centrifuging at 54,000 g for 30 min at 20°C using a swing out rotor, the pellet was resuspended in lysis buffer containing taxol and GTP. To extract the MAPs, the concentration of NaCl was adjusted to 0.4 M, and the samples were incubated for 30 min at 37°C. Microtubules were pelleted by centrifugation at top speed on a microcentrifuge for 30 min; the supernatant is considered to be the MAP fraction.
Overlay Assays
Proteins were blotted to PVDF membranes (Amersham Life Sciences, Pittsburgh, PA) after SDS-PAGE. Membranes were blocked with TBST (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk for 1 h, washed for 15 min in TBST plus 1% nonfat dry milk, and washed for 15 min in PEMF (0.1 mM Pipes/NaOH, pH 6.6, 1 mM EGTA, 1 mM MgSO4, 25 mM NaF) buffer plus 1 mM GTP. The blots were then incubated overnight at room temperature with 5 µg/ml Drosophila tubulin (purified as described) in PEMF supplemented with 1 mM GTP and 20 mM taxol. The bound microtubules were detected by standard Western blot procedures using the monoclonal antibody Bx69 diluted five times.
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insertion vector 1149. The chromosomal origins of these clones were confirmed by in situ hybridization, before using them to screen phage and cosmid genomic libraries (see Materials and Methods). A resulting chromosome walk of 130 kb was carried out and clones correlated with the polytene chromosome map by in situ hybridization. The distal breakpoint of In(3R)Ubx7LLatsR, which does not uncover asp and defines the proximal boundary of the region known to contain asp, was mapped to a 5.8-kb BamHI fragment at the extreme proximal end of the walk. The distal end of the chromosome walk maps to the interband between 96A21-25 and 96B1-10. Subsequently, we found that a cytologically invisible deficiency Df(3R)Hd1 uncovers not only the nearby zygotic embryonic lethal tolloid (tld), as reported by Shimell et al. (1991), but also asp.
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4 kb that removes the entire tld transcription unit and 1 kb of the 5' end of the gene encoding the 6.5kb transcript. Shimell et al. (1991) showed that tld can be rescued by two constructs, pMBO1366 and pMBO1367. Although pMBO1366 rescues the early embryonic zygotic lethality of tld68–62, the rescued flies exhibit cuticular defects. As such defects are associated with asp, it seemed that tld68–62 could be a tld asp double mutant. Consequently we tested whether these same transformants would rescue the recessive lethality of asp1. pMBO1366 contains tld but only the 5' end of the candidate asp gene, whereas pMBO1367 contains both transcription units (see Materials and Methods; Fig. 1). We found that the pMBO1367 rescues the lethality of asp1 mutants while pMBO1366 does not. Taken together, these data confirm the identity of the 6.5-kb transcription unit as asp.
Furthermore, this gene has a developmental pattern of expression typical of many genes essential for the cell cycle whose products have to be maternally provided to the embryo. We used EcoRI–EcoRI or NcoI–EcoRI fragments from the 6.5-kb cDNA clone p6a as hybridization probes on developmental Northern blots. One transcript of 6.5 kb was readily observed (Fig. 2) in mRNA prepared from embryos and females (Fig. 2, lanes 1, 2, and 6) but was present at greatly reduced levels in larval and adult male mRNA (Fig. 2, lanes 3–5 and 7). When a SalI– NotI fragment from the plasmid pMBO1367, encompassing around 14 kb upstream of the NotI site on asp, was used as a probe, we observed the same 6.5-kb transcript plus one 3.8-kb transcript in embryonic mRNA corresponding to tld (Shimell et al., 1991) and a 5.2-kb transcript presumably corresponding to the recently described tolkin gene (Finelli et al., 1995; and data not shown).
asp Encodes a Highly Basic Protein with Putative Actin and Calmodulin Binding Domains
The 6.5-kb asp cDNA encodes a predicted polypeptide of 1,863–amino acid residues that has no homologues in the GenBank/EMBL/DDBJ database (Fig. 3 A). The asp protein is predominantly hydrophilic and strikingly basic, having a calculated pI of 10.8. Its secondary structure is predicted to be mostly 
-helical. Analysis of the protein using the COILS program shows that short stretches of amino acids near the COOH terminus have the potential to form a coiled coil. There is a small sequence lying between residues 848 and 870 that has significant similarity to the core actin binding domain of a number of actin binding proteins, such as -actinin (Noegel et al., 1987; Blanchard et al., 1989), fimbrin, spectrin, dystrophin (de Arruda et al., 1990; Matsudaira, 1991), and the Dictyostelium discoideum ABP120 (Bresnick et al. 1990; Fig. 3 B). These proteins either bundle actin filaments together (for example -actinin) or attach actin filaments to other cellular structures. A second sequence lying between residues 938 and 968 corresponds to the conserved calmodulin binding (IQ) motif (Cheney and Mooseker, 1992) present in neuromodulin, a neuron-specific membrane-associated protein (Chapman et al., 1991); neurogranin, a neuron specific protein kinase C substrate (Baudier et al., 1991); the igloo gene product, a calmodulin-binding protein from the Drosophila central nervous system (Neel and Young, 1994); and in the "neck" regions of most forms of conventional and nonconventional myosin (for review see Cheney and Mooseker, 1992; Fig. 3 C). In addition Asp shows six consensus sites for phosphorylation by p34cdc2 and four consensus sites for phosphorylation by MAP kinase. Interestingly, these are all clustered in the NH2-terminal third of the molecule.
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220 kD in immunoblots of third instar larval brains (Fig. 5). To confirm that Rb3133 reacts specifically with the Asp protein, brains from larvae of the transgenic strain 1366 (see Materials and Methods) were used in Western blot analysis (Fig. 5, lane 2). Flies transformed with pMBO1366 are expected to synthesize two forms of the Asp protein: full length protein of 220 kD, derived from the endogenous copy of asp at 96A and a truncated form derived from the transgene. As expected, the serum detects the 220-kD wild-type asp protein plus a 124-kD polypeptide corresponding to the COOH-terminal truncated form of the asp protein encoded by plasmid pMBO1366. When extracts from brains of asp1 homozygous larvae were analyzed (Fig. 5, lane 3), the 220-kD protein is no longer observed, but the serum labels a polypeptide of 130 kD. As the polyclonal antibodies were raised against the NH2 terminus of the Asp protein, it seems that the asp1 mutation results in a COOH terminus truncation or in an internal deletion of the Asp protein.
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Discussion |
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Asp is the first metazoan MAP to be identified through a genetic approach, and it is gratifying that the naming of the gene by Ripoll et al. (1985) was predictive of the localization and function of the protein. The protein is highly basic, and in its central region lies a putative actin-binding motif. Basic regions within other MAPs play a significant part in conferring the ability to bind microtubules. The Asp protein is no exception to this rule in that at least one segment of this basic molecule, a polypeptide comprised of the 512 NH2-terminal amino acids, can be shown to bind microtubules in vitro. The central region of the protein binds microtubules poorly. Unfortunately, we have been unable to test the microtubule binding ability of the COOH-terminal region, as this segment of the protein appears unstable in both bacterial and insect cell expression systems.
It is instructive to compare the domain organization and microtubule binding properties of Asp with other MAPs that have been characterized. These are principally of mammalian origin, and most have been purified from brain as proteins associated with the stable microtubules of the axons and dendrites of neurons. In some cases, these proteins may be expressed specifically in brain. However, in many cases their presence in other tissues is being confirmed. MAP2, for example, is predominantly found in brain but has recently been shown to be present in rat testes (Loveland et al., 1996). In general, MAPs bind to microtubules to increase their stability. Studies of MAP2C in vivo, for example, show that it bundles and stabilizes microtubules in cultured fibroblasts (Umeyama et al., 1993). Moreover, in vitro studies show that MAP2 can decrease the frequency of transitions between the growth and shortening of microtubules leading to net microtubule growth (Kowalski and Williams, 1993; Itoh and Hotani, 1994). Like several other MAPs, MAP2 has a short "promoter" domain, which binds to microtubules to promote assembly, and a "projection" domain thought to interact with other cytoskeletal elements. Fellous et al. (1994) have digested MAP2 with chymotrypsin to remove the projection domain and find that the residual promoter domain differs from the full length protein in that it binds microtubules to organize them into clusters of spirals. Moreover, assembly of microtubules in the presence of the promoter domain alone is more resistant to certain depolymerizing drugs than assembly in the presence of the full length protein. Thus the projection domain would appear to modify the interactions of MAP2 with microtubules in their assembly. The Asp protein may be seen as being structurally similar and functionally analogous, having an NH2-terminal microtubule binding domain and central and COOH-terminal regions with the potential to interact with other cytoskeletal components.
It may be not only that microtubule dynamics are affected by the asp mutation but possible that the function of other MAPs, in particular motor molecules, could be impaired if the mutant protein can still bind microtubules. The binding of MAP2, for example, is known to inhibit the motility of kinesin and cytoplasmic dynein, but this is reversed by phosphorylation of the protein (Lopez and Sheetz, 1995). This specifically requires a cAMP-dependent kinase that copurifies with MAP2, and it is not affected by phosphorylation with p34cdc2 kinase, mitogen activated protein kinase, or the NIMA mitotic kinase. In contrast, phosphorylation of MAP4 by p34cdc2 kinase abolishes its microtubule stabilizing activity, interestingly without altering its ability to bind microtubules (Ookata et al., 1995). MAP4 is the major MAP of many mammalian cell lines and is present along the length of both interphase and mitotic microtubules (Bulinski and Borisy, 1980; DeBrabander et al., 1981). There is evidence that MAP4 mediates the association of cyclin B with microtubules in both the starfish oocyte and in cultured mammalian cells (Ookata et al., 1992, 1993, 1995). It may therefore provide a general mechanism for destabilizing interphase microtubules upon entry into mitosis. Other MAPs could also have such a role, and as with the kinesin-like proteins, MAPs could be functionally redundant. A 230-kD protein, XMAP 230, has been purified from Xenopus oocytes and found to be present in all dividing cells (Andersen et al., 1995). It is localized to microtubules in interphase but dissociates from them upon entry into mitosis, to reassociate later in the mitotic cycle. It is phosphorylated by mitotic extracts whereupon it has a reduced affinity for microtubules. Upon binding it increases microtubule growth rate, decreases the rate of shrinking, and suppresses microtubule loss by "catastrophe." It thus stabilizes interphase microtubules and can locally modify microtubule behavior during mitosis. As the Asp protein has numerous sites for phosphorylation by p34cdc2 and mitogen-activated protein kinase, it is not inconceivable that cyclical phosphorylation could affect its properties throughout the mitotic cycle.
The presence of wild-type Asp protein in the polar regions of the spindle points to this being the site of its action and suggests that it may play a crucial role in regulating the dramatic changes of microtubule dynamics upon the entry into mitosis. Its presence in the astral microtubules may be important to mediate interactions between microtubules and the actin cytoskeleton. This function is suggested by the actin-binding motif in the central domain of the protein. This motif is homologous to ones seen in a number of actin bundling or gelation factors. These include -actinin (Noegel et al., 1987; Blanchard et al., 1989), a filamentous actin cross-linking protein found in stress fibers and adhesion plaques in nonmuscle cells and in Z-discs in muscle cells; it has a single actin binding motif and can cross link actin filaments into a homodimer. Single actin binding motifs are also found on dystrophin and spectrin (de Arruda et al., 1990; Matsudaira, 1991). Fimbrin, an actin-associated protein found in microvilli and filopodia, on the other hand, has a duplicated actin binding motif that enables it to bundle actin filaments as a monomer (de Arruda et al., 1990). It has homology to plastins, cytoplasmic actin binding proteins, and the protein encoded by the yeast gene ABP7 or Sac6p (Adams et al., 1995). The association of microtubule and microfilament networks is nowhere more obvious than in the vicinity of centrosomally nucleated asters. In the syncytial blastoderm Drosophila embryo, for example, the actin caps that form above interphase nuclei are positioned by the centrosome and its associated microtubules. Moreover, the spatial coordination of mitosis and cytokinesis requires the contractile actin ring that will dictate the cleavage furrow to be correctly positioned with respect to the spindle. Classical experiments with echinoderm eggs showed that moving the spindle by micromanipulation resulted in the corresponding repositioning of the cleavage furrow. However, the experimental manipulation of such embryos to produce two mitotic spindles within a single cell led to the generation of a third cleavage furrow between the asters of the juxtaposed spindles (for review see Rappaport, 1986a,b). The nature of the molecular "signal" that positions the actin ring with respect to the asters is unknown, but it is possible that a molecule such as the Asp protein could fulfill this role.
The presence of a potential calmodulin binding site in the central region of the molecule implies that the Asp protein may be responsive to fluxes in calcium ions known to occur in the polar regions of the spindle. Preliminary experiments show that the motif present in Asp enables it to bind to calmodulin in a Ca2+-dependent manner (data not shown). Calmodulin is a member of the EF hand superfamily of proteins. There is strong evidence for a direct role for two members of this family in spindle pole body (SPB) function in budding yeast. Calmodulin itself has been shown by both genetic and molecular studies (Geiser et al., 1993; Stirling et al., 1994, 1996; Sundberg et al., 1996) to be essential for spindle function as a result of its interaction with the COOH terminus of Spc110p, an essential protein with a large coiled-coil domain that provides a spacer between the central and inner plaques of the SPB (Kilmartin, 1993). The second EF hand protein localized to the SPB is the product of the CDC31 gene, which associates with the KAR1 gene product to form a complex (Spang et al., 1993, 1995; Biggens and Rose, 1994; Vallen et al., 1994). Roles for these proteins in spindle function are likely to be conserved. Calmodulin is the major intracellular Ca2+ receptor and is involved in many cellular processes; it is known to be localized to the microtubules of metazoan spindles (e.g., Stemple et al., 1988), and it appears to be important in regulating the function of its dependent kinase and phosphatase in the metaphase– anaphase transition and in spindle behavior (Morin et al., 1994; Yoshida et al., 1994). CDC31 is the yeast homologue of centrin, a protein first identified in the Chlamydomonas basal body and subsequently found to be a universal component of centrosomes in animal cells (for review see Salisbury, 1995). There are therefore a large number of potential ways in which Asp protein function might be mediated through an association with one of these EF hand proteins.
It is evident that many questions are thrown open by our finding that the Asp protein is a MAP containing potential actin and calmodulin binding motifs. However, our work has now generated the molecular tools with which we can begin to address some of the above issues.
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Acknowledgments |
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This work was supported by research grants from Science and Engineering Research Council, Medical Research Council, Cancer Research Campaign, and the Human Capital and Mobility Programme of the European Union. Thomas Howard is grateful for a studentship from BBSRC and Maria do Carmo Avides for an European Molecular Biology Laboratory Long-Term Fellowship.
Submitted: 5 October 1996
Revised: 30 January 1997
1. Abbreviations used in this paper: asp, abnormal spindle; spb, spindle pole body; tld, tolloid.
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