Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10285-10289, October 1994 Cell Biology Ankyrin and ,-spectrin accumulate independently of a-spectrin in Dro8ophila RONALD R. DUBREUIL* AND JIANQING YU Department of Pharmacological and Physiological Sciences and The Committee on Cell Physiology, University of Chicago, 947 East 58th Street, Mail Code 0926, Chicago, IL 60637 Communicated by Hewson Swit, June 30, 1994 ABSTRACT We report the identification and Initial char- skeleton in nucleated cells. Studies in Drosophila have so far acterization ofDrosophila nelanogaster ankyrin. Oligonucleo- shown that three membrane skeleton proteins originally tide primers based on the spctrin-binding domain of human identified in the mammalian erythrocyte are conserved in brain ankyrin were used to amplify Drosophila genomic DNA. evolution and have essential roles in development. The hts A cloned 184-bp PCR product was used to Isolate Drosophila protein is a homolog of adducin that is likely to be associated ankyrin cDNAs. Ankyrin cDNA probes detected a 5.5-kb with actin in ring canals of the developing oocyte (14). transcrptfrom embryonic poly(A)+ RNA and a single polytene Defects in hts block egg development. D4.1 is a homolog of chromosome locus (1O1F-102A). The cDNA sequence encodes protein 4.1 (15) that is encoded by the essential coracle gene. a 170-kDa protein that is 53% identical to human brain Defects in D4.1 block dorsal closure during embryonic de- ankyrin (Ank2). Antibodies directed against a recombinant velopment and suppress the dominant eye defect in epidermal fragment of Drosophila ankyrin reacted with a 170-kDa poly- growth factor receptor mutants. peptide from Drosophila embryos, larvae, S2 cells, and adult Drosophila spectrin is also conserved in its structure, flies. The ankyrin antibody immunopripitated a- and composition, and actin-binding activity (16). Mutations ofthe ,-spectrin with ankyrin in detergent extracts of Drosophila a subunit are lethal during larval development (17). Surpris- embryo membranes. Antibodies against Drosophila ankyrin, ingly, some aspects of development do not appear to be a-spectrin and «spectri were used to detect these proteins in grossly affected by diminished levels of a-spectrin. Mutant wild-type and a-sin mutant larvae. a-Spectrin levels larvae, which retain only a small quantity of maternally were greatly diminished in mutant larvae, but levels ofankyrin derived a-spectrin, are initially able to crawl, feed, and and -spectrin were indis hable from wild type. The respond to tactile stimuli. They appear normal in overall form persistence of ankyrin and P-spectrin may explain the rela- and development ofmajor larval structures, but they become tively mild phenotype of a-spectrin mutants during early lethargic and die before the first larval molt. Close inspection Drosophila development. of the larval gut reveals a significant effect of a-spectrin mutations on the shape and adhesion ofone class ofepithelial The protein ankyrin provides a link between integral mem- cells (17). brane proteins and the spectrin-based membrane skeleton We have initiated studies of ankyrin in Drosophila to (1). That link appears to be important to the mechanical further dissect the cellular roles of the membrane skeleton. properties of the plasma membrane in mammalian erythro- An ankyrin gene was identified by amplification of genomic cytes. Defects in ankyrin result in membrane fragility, a DNA with oligonucleotide primers based on a conserved decrease in the number of circulating mature erythrocytes, region ofmammalian brain ankyrin.t We used a collection of and consequent anemia (2, 3). The membrane defect results antibodies against this ankyrin homolog and the spectrin from the failure of spectrin and other proteins to assemble subunits of Drosophila to examine the fate of the membrane properly without ankyrin as an attachment site. Defects in skeleton in the absence of a-spectrin synthesis. spectrin, which produce a similar phenotype, do not affect the accumulation of membrane-bound ankyrin (4). In contrast to its interaction with a single major transmem- MATERIALS AND METHODS brane protein in erythrocytes (5), ankyrin interacts with Amplification of Ankyrin DNA. Genomic DNA from adult several different proteins in nucleated cells. Coimmunopre- Drosophila melanogaster was used in PCRs with synthetic cipitations, cosedimentation, and in vitro binding assays have primers based on the human brain ankyrin 2 sequence (18): been used to demonstrate interactions between ankyrin and AnkAl, CCA CTT TAT CAT CAG TCA TGC; and AnkS1, the sodium pump (6), sodium channels (7, 8), a nonerythroid AAC GTG TCT GCC AGG TTC TGG. Reactions were anion-exchange protein (AE3; ref. 9), a cell adhesion mole- carried out in a MiniCycler (MJ Research, Cambridge, MA) cule from brain (ABGP1; ref. 10), and others (reviewed in ref. for 40 cycles (940C for 1 min, 450C for 2 min, 720C for 1 min) 11). Each of these interactions may simply attach the spec- with Taq DNA polymerase (Perkin-Elmer/Cetus). trin-based membrane skeleton to the plasma membrane. Isolation of Ankyrin cDNA. A 0- to 4-hr Drosophila mela- Alternatively, it has been proposed that ankyrin can act in the nogaster embryo cDNA library (19) was screened with the fly opposite direction: to capture and stabilize membrane pro- ankyrin PCR product by standard methods (20). The probe teins in specific membrane domains defined by the membrane was amplified from cloned plasmid DNA and hybridized at skeleton (12, 13). But it has not been possible to directly test high stringency under conditions described by O'Neill and the role of ankyrin in membrane domain segregation. Belote (21). Additional cDNAs were recovered from a size- Genetic studies have been important in unraveling the roles fractionatedlarvallibrary(generouslyprovidedbyCarlThum- of membrane skeleton proteins in the erythrocyte, and they mel, University ofUtah), using the A fragment ofclone A411 are now contributing to our understanding of the membrane as probe (Fig. 1). The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L35601). 10285 Downloaded by guest on September 24, 2021 10286 Cell Biology: Dubreuil and Yu Proc. Natl. Acad. Sci. USA 91 (1994) All RESULTS A411 A631 Synthetic oligonucleotide primers based on the conserved Flank spectrin-binding domain ofthe human ANK2 gene were used A631 B in the PCR with Drosophila genomic DNA. A 184-bp product E X SB E E pNB-40 [ 1I : II (Flank, Fig. 1) was amplified, cloned in pBluescript, and vectorr I I sequenced. The predicted amino acid sequence of the fly 1 kL ankyrin fragment was -40%o identical to the sequences of mammalian ankyrins (18, 30). FIG. 1. Drosophila ankyrin cDNA clones. Primers based on the Isolation and Characterization of Drosophila Ankyrin sequence ofhuman brain ankyrin 2 were incubated with fly genomic cDNA. The cloned fly ankyrin sequence was used as a probe DNA to produce the fly ankyrin (Flank) PCR product. Ankyrin cDNAs were isolated from embryonic (A411 and A631) and imaginal to screen libraries of Drosophila embryonic cDNA. The disc libraries (All). A composite restriction map of the cDNAs is probe failed to hybridize with fly DNA under standard shown at the bottom (E, EcoRl; S, Stu I; B, BamHI; X, Xba I). Dots high-temperature conditions, but several cDNA clones were mark the boundaries of clone A411 probes. isolated from a 0- to 4-hr embryonic cDNA library after hybridization in 50%o formamide at 42TC. Additional clones Southern and Northern Blots and Polytene Hybridizations. from the 5' region of the coding sequence were recovered Blots of fly genomic DNA and RNA were prepared by from a size-selected larval cDNA library by using the A standard methods (20). Biotin-labeled probes (Polar-Plex fragment of clone A411 as probe. Overlapping regions of labeling kit, New England Biolabs) were used in Southern these cDNAs produced identical digestion patterns with blots (1 fly per lane). A 32P-labeled C fragment of cDNA 411 several restriction enzymes, suggesting that they are all (Fig. 1) was used in Northern blots [8 pg of poly(A)+ RNA partial cDNA products derived from a single class ofankyrin per lane]. Polytene chromosomes from Oregon-R Drosophila transcript (Fig. 1). larvae were prepared by standard methods (22). Southern blots of Drosophila genomic DNA were probed DNA Sequencing. The cDNA sequence was determined on with the EcoRI A fragment of cDNA A411 (Fig. 2A). The both strands by using a Sequenase kit (United States Bio- probe spans a BamHI restriction site in the cDNA sequence chemical) and synthetic oligonucleotide primers. A compos- and therefore detects two bands in BamHI-digested genomic ite sequence was assembled by using cDNA clones All and DNA (lane B). An Xba I-Stu I fragment (A411 C fragment in A631 (Fig. 1). Sequence assembly and dot-plot comparisons Fig. 1) from upstream of the BamHI site detects only the were generated by using the Genetics Computer Group lower 5.8-kb band, and an EcoRV fragment probe from (Madison, WI) programs (23), as previously described (24). downstream ofthe BamHI site detects only the upper >10 kb Production of Antibodies. Recombinant fragments of fly band (not shown). The A411 A probe does not span an EcoRP ankyrin and 3-spectrin were produced as fusions with glu- site in the cDNA sequence, but it still detects two bands (2.8 tathione S-transferase and used to generate rabbit antibodies. kb and >10 kb) in EcoRI-digested genomic DNA (lane E). The B fiagment of ankyrin cDNA clone A631 (Fig. 1) was There may be an intron in the ankyrin gene that contains an expressed in the pGSTag vector (25).