INTRODUCTION

Extracellular matrix (ECM) arises during embryogenesis andprobably has two major functions in development. First, it isthe macromolecular environment of differentiating cells andgoverns the access of cells to signal input. Secondly, itprovides mechanical continuity between emerging tissues.Interactions between individual ECM protein ligands and cellsurface receptors need to be studied in animals to understandtheir development. Here we report on the interaction betweena new

Drosophila ECM protein, tiggrin, and

αPS2βPS integrins.Integrins constitute an important group of ECM receptors

(reviewed by Hynes, 1992). The major Drosophila integrins(reviewed by Brown, 1993; Bunch and Brower, 1993), PS1(αPS1βPS) and PS2 (αPS2βPS), were discovered as positionspecific (PS) antigens (Wilcox et al., 1981; Brower et al.,1984). Their similarity to vertebrate integrins was recognized,and the sequence homologies of their subunits with vertebrateα and β chains have been established (Bogaert et al., 1987;Leptin et al., 1987; MacKrell et al., 1988; Wehrli et al., 1993).

Genetic analyses of mutations in the genes encoding theαPS2 and βPS subunits suggest that a major function of theDrosophila integrins is to stabilize strong attachments betweencells (Wright, 1960; Brower and Jaffe, 1989; Wilcox et al.,1989; Zusman et al., 1990; 1993; Brabant and Brower, 1993;Fristrom et al., 1993), and cytological observations indicate

that these associations are mediated by ECM components (e.g.Newman and Wright, 1981). A striking example of this occursin the embryonic lethal mutant myospheroid, which lacks thecommon βPS chain: its muscle insertions rupture on musclecontraction (Wright, 1960). This is consistent with the corre-sponding absence of αPS2βPS integrin from the ends ofmyotubes, where it is normally concentrated, and of αPS1βPSintegrin from the apposed tendon cells (Leptin et al., 1989).

We have found a new protein, tiggrin, that is a componentof ECM at muscle insertions (apodemes). The concentration oftiggrin at these segmentally repeated sites gives embryosstained with anti-tiggrin antibodies the striped appearanceassociated with tigger, a tiger-like entity (Milne, 1938). OtherECM proteins that have been identified at Drosophila muscleinsertions are collagen IV, papilin and glutactin (Fessler andFessler, 1989). Transcripts of tena, a member of the tenascinfamily, are also found in cells located at apodemes (Baum-gartner and Chiquet-Ehrismann, 1993).

In vitro experiments on substrates of individual, purifiedECM proteins showed that primary Drosophila embryo cellsand established Drosophila cell lines that specifically expressαPS2βPS integrins can spread on two vertebrate integrinligands, fibronectin and vitronectin (Volk et al., 1990; Hiranoet al., 1991; Bunch and Brower, 1992; Gullberg et al., 1994;Zavortink et al., 1993). The cell attachment sequence Arg-Gly-Asp (RGD; Ruoslahti and Pierschbacher, 1987; Yamada,

1747Development 120, 1747-1758 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

Genetic and other studies of

Drosophila integrins haveimplicated these extracellular matrix receptors in variousmorphogenetic events, but identification of their endoge-nous ligands has been elusive. We report the biochemicalpurification and cloning of tiggrin, a novel extracellularmatrix protein from Drosophila. This 255×103 Mr polypep-tide contains the potential integrin recognition sequenceArg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 aminoacid motif. The tiggrin gene is at chromosome locus 26D1-2 and is expressed by embryonic hemocytes and fat body

cells. Tiggrin protein is detected in matrices, especially atmuscle attachment sites that also strongly expressintegrins. Tiggrin-coated surfaces support primary embryocell culture and provide excellent substrates for αPS2βPSintegrin-mediated cell spreading. Soluble RGD-peptidesinhibit this cell spreading.

Key words: Drosophila, Embryogenesis, Extracellular matrixproteins, Integrins, Muscle-development

SUMMARY

Tiggrin, a novel

Drosophila extracellular matrix protein that functions as a

ligand for Drosophila αPS2βPS integrins

Frances J. Fogerty1,*, Liselotte I. Fessler1, Thomas A. Bunch2, Yifah Yaron1, Carol G. Parker1, Robert E.Nelson1, Danny L. Brower2,3, Donald Gullberg1,† and John H. Fessler1,‡

1Department of Biology and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90024-1606,USA2Department of Molecular and Cellular Biology, 3Department of Biochemistry, University of Arizona, Tucson, AZ 85721, USA*Present address: Department of Medicine, University of Wisconsin, Madison, WI 53706-1532, USA†Present address: Department of Animal Physiology, BMC, University of Uppsala, 7124 Uppsala, Sweden‡Author for correspondence

1748

1991) that occurs in fibronectin and vitronectin is important forthis interaction with αPS2βPS integrin, as spreading is inhibitedby added soluble RGD-peptides (Bunch and Brower, 1992;Zavortink et al., 1993). In contrast, several Drosophila ECMproteins that were potential candidate ligands failed to supportαPS2βPS integrin-mediated cell spreading, namely Drosophilacollagen IV, papilin, laminin and glutactin (Volk et al., 1990;Bunch and Brower, 1992).

The search for a Drosophila αPS2βPS integrin ligand atmuscle insertions led to tiggrin, which is a good candidate fortwo reasons. First, it occurs at several sites where integrins areconcentrated, including muscle apodemes and muscle Z-bands(see Bogaert et al., 1987; Leptin et al., 1989; Volk et al., 1990;Wehrli et al., 1993 for integrin distribution around muscles).Secondly, the potential cell attachment sequence RGD occursin the amino acid sequence that we deduced for tiggrin. Ourresults of in vitro cell spreading assays strongly implicatetiggrin as the first reported ligand of Drosophila integrins. Theprotein is of further interest because more than half of itconsists of a novel repeat motif.

MATERIALS AND METHODS

Purification and characterization of tiggrinThe Drosophila Kc-derived 167 cell line was grown as a monolayerin D22 medium with 2% heat inactivated FCS and the conditionedmedium was collected at 1 or 2 day intervals for 1 week. After addingprotease inhibitors the medium was clarified, and the proteins pre-cipitated by 40% (NH4)2SO4 saturation and were partly separated bysucrose gradient velocity sedimentation as described previously(Fessler et al., 1994). Fractions containing tiggrin were chro-matographed on a Mono Q anion exchange column (Pharmacia LKBBiotechnology) at 4oC in 30 mM Tris-HCl, pH 8.0, 10 mM EDTA,0.3 M sucrose, 1 mM Chaps or 0.05% Triton X-100, 3 mM NaN3(Mono Q buffer). Bound proteins were eluted with a 0-0.5 M NaClgradient. Tiggrin was further purified by gel exclusion chromatogra-phy on a Superose 6 column (Pharmacia LKB Biotechnology) inMono Q buffer containing 0.15 M NaCl. Deglycosylation withPeptide:N-glycosidase F (PNGase F) (New England Biolabs) wascarried out according to the manufacturer’s protocol.

The corrected sedimentation coefficient, s20,W, of tiggrin, purifiedin the absence of Chaps, was determined on a 5-20% sucrose gradientin 150 mM NaCl, 50 mM Tris-HCl, 0.1% Triton X-100, pH 7.5 (TBS)by a modification by Fessler and Fessler (1974). The hydrodynamicfrictional ratio, f/fo = 2.17, was calculated from s20,w and Mr =255×103, assuming the usual values of partial specific volume v̄ = 0.73ml/g and hydration 0.1. The corresponding equivalent prolateellipsoid of revolution, or rod-like particle, is 70 nm long and has a2.9 nm diameter (van Holde, 1985). For a molecule consisting of 2tiggrin chains the predicted length and diameter are 180 nm and 3.1nm. Variations of the assumed v̄ and hydration values have little effecton these results.

Protein sequencingTo obtain the N-terminal sequence, reduced tiggrin was elec-trophoresed on an SDS 4.5% polyacrylamide gel, electroblotted ontoImmobilon-P membrane (Millipore) (LeGendre and Matsudaira,1988) and submitted for microsequencing (Hewick et al., 1981). Toobtain the internal amino acid sequence, in situ CNBr cleavage (Scottet al., 1988) was performed on a tiggrin band cut out of a SDS 4.5%polyacrylamide gel (Kurth and Stoffel, 1990). The resulting peptideswere separated by SDS-10%-PAGE, electroblotted and microse-quenced.

Antibody preparationMice were immunized with purified non-reduced tiggrin, or with thereduced RGD-containing fusion protein, excised from a SDS poly-acrylamide gel. Affinity-purified antibody was prepared by incubat-ing nitrocellulose-bound tiggrin, or the RGD-containing fusionprotein, with the polyclonal antiserum (Robinson et al., 1988).

Western blotting of Drosophila embryo detergent extractsOregon R Drosophila embryos were collected, kept at 25˚C, andextracts were made as described (Fessler et al., 1994). Urea was addedto the reduced, sonicated and heated homogenate to 6 M and anyremaining insoluble material was removed by centrifugation. Proteinconcentration was determined using the BioRad protein assay.Samples of 100 µg protein were electrophoresed in an SDS 4.5%polyacrylamide gel and electroblotted to nitrocellulose. Themembrane was blocked, reacted with antiserum to tiggrin, washed,and reacted with 125I-Protein A (Amersham) as described (Fessler etal., 1994).

cDNA library screening and DNA sequencingStandard protocols were used (Sambrook et al., 1989). Purifiedpoly(A)+ RNA (Kusche-Gullberg et al., 1992) isolated fromDrosophila Kc 167 cells was used to prepare a cDNA expressionlibrary in λ Zap (Blumberg et al., 1992). A portion of the unampli-fied library (3.6×105 pfu) was plated on E. coli strain YS1 andscreened with affinity-purified anti-tiggrin antibodies. Five out of 18immunopositive clones contained overlapping inserts and werederived from the 3′ end of the mRNA since two of them encodedpoly(A) tails. A 32P-labelled, 5′ fragment of the clone that extendedthe farthest in the 5′ direction was used to select longer clones fromthe pNB40 12-24 hour Drosophila embryo library (Brown andKafatos, 1988). This library, transformed into E. coli XL1-Blue, wasplated on 18 150 mm plates at a density of 14,000 cfu per LB/ampplates and grown overnight at 37˚C. Individual plates were scrapedinto 3 ml TB/amp. A 700 µl aliquot was used to prepare a frozenglycerol stock from each plate and the remainder was used to prepareplasmid DNA. The plasmid DNA (10 µg) from each plate was lin-earized with NotI and a Southern blot was screened. From the subli-brary that contained the largest clone a 6,895 bp clone containing theentire protein coding region was isolated and sequenced.

Selected restriction fragments were subcloned into eitherM13mp18, M13mp19 or Phagescript (Stratagene) and sequenced bythe dideoxy chain termination method using α-35S-dATP and theSequenase sequencing kit (US Biochemicals). The sequence wasdetermined on both strands and across all restriction sites used in sub-cloning. The resulting sequence data were compiled and assembledusing the Staden computer package (Staden, 1986). Additionalsequence analysis was performed using the UWGCG computerpackage. The Prot 99 database is a combination of databasesSwissProt (release 23), PIR (release 34) and Genbank (release 74).

Tiggrin fusion proteinA PstI cDNA fragment encoding 270 amino acids (residues 1,891-2,161) that include the RGD cell attachment sequence was cloned intothe pTrcHisC bacterial expression vector (Xpress SystemTM, Invitro-gen). A control plasmid contained the above cDNA subcloned in thereverse orientation and encoded 90 amino acids. Inclusion bodieswere solubilized in 8 M urea, 30 mM Tris, pH 8.0, 1% Triton X-100,1 mM EDTA, 1 mM PMSF (1/50 the original culture volume). Thefusion proteins were slowly renatured by stepwise dialysis into TBS.Some of the tiggrin fusion protein was further purified by immobi-lized metal affinity chromatography with Invitrogen’s ProBondTM

resin. Both affinity-purified and urea-solubilized tiggrin fusionproteins, dialyzed into PBS, gave the same result in cell attachmentassays.

F. J. Fogerty and others

1749Tiggrin,

Drosophila integrin ligand

RNA isolation and northern analysisTotal RNA was isolated from Drosophila embryos, kept at 25˚C, at2 hour intervals of embryogenesis, by the guanidine thiocyanatemethod and northern analysis was done as described (Kusche-Gullberg et al., 1992). Northern blots were probed with 32P-labeledRNAs synthesized with T7 RNA polymerase and an RNA transcrip-tion kit (Stratagene). The template encoding the antisense probecontained tiggrin cDNA sequence nucleotides 960-3250.

In situ hybridization to embryos and Drosophila polytenechromosomesIn situ hybridization to embryos was performed by the method ofTautz and Pfeifle (1989) as modified by Dr Bruce A. Edgar (personalcommunication) using a digoxigenin-labelled probe containing tiggrincDNA sequence corresponding to nucleotides 363-555. In situ hybrid-ization to polytene chromosomes was done according to the methodof Langer-Safer et al. (1982) using a biotinylated probe containingtiggrin sequence nucleotides 5520-6895.

Immunostaining of embryosWhole mounts of Oregon R or myospheroidXG43 embryos wereimmunostained essentially as described by LeMotte et al. (1989). 10µm cryostat sections were fixed for 3 minutes in 2% paraformalde-hyde, 1% Triton X-100 and immunostained. The primary antibodieswere mouse anti-tiggrin, anti-RGD-containing fusion protein orpreimmune serum and rabbit anti-collagen (Lunstrum et al., 1988),rabbit anti-ßPS (Gullberg and Fessler unpublished data) and mousemonoclonal antibodies to βPS (Brower et al., 1984). The secondaryantibodies were rhodamine or fluorescein-labelled anti-mouse IgG oranti-rabbit IgG (Jackson Immune Research) and biotinylated anti-mouse IgG and the biotin/avidin-peroxidase system (Vector Labs).

Cell culture and cell spreading assaysThe cells used for cell spreading measurements and the assay methodhave been described previously (Bunch and Brower, 1992; Zavortinket al., 1993). Briefly, Schneider line 2 (S2) Drosophila cells(Schneider, 1972) were used that had been cotransfected with cDNAscoding for integrin subunits, under the regulation of the HSP70 heatshock promoter, and the bacterial dihydrofolate reductase gene. Afterremoval of extracellular proteins with dispase/collagenase the washedcells were heat shocked to induce the βPS and αPS2 transgenes’expression. The cooled, washed cells were resuspended at 2-4×105

cells/ml in M3 medium containing 2 mg/ml BSA and only 68 µM Ca2+

(1/100 the normal M3 medium levels of Ca2+). This lower Ca2+ levelin this medium is more favorable to αPS2βPS integrin mediated cellspreading (Zavortink et al., 1993). 96-well microtiter dishes werecoated overnight, at 4˚C, with 5 µg/ml or 1 µg/ml tiggrin in PBS, orwith the equivalent concentrations of fusion proteins. Before use, thetiggrin-containing solution was removed and the wells were blockedwith 10% non-fat Carnation milk in PBS for 1-1.5 hours at 22˚C. Thissolution was removed and the wells rinsed twice in Robb’s salineminus Ca2+/Mg2+ and once with M3 medium minus Ca2+/Mg2+. Forthe RGD inhibition studies, after recovery from heat shock cells werediluted to a cell density of 4×105/ml and diluted 50% into microtiterwells containing M3 medium with various concentrations of the testpeptides. For the antibody blocking experiments, purified IgG fractionswere added to the cells, at a final concentration of 10 µg/ml, just priorto plating. For all spreading experiments, cells were fixed 3-5 hoursafter heat shock. Three microscope fields from each well were pho-tographed and the number of round and spread cells was scored blindlyto determine the percentage of spread cells. An average of the threefields was obtained for each experiment. The numbers given in thefigures represent the averages (and standard errors) of at least threeseparate experiments. Primary Drosophila embryo cell cultures (Volket al., 1990) were made on coverslips coated with 30 or 60 µg/mltiggrin or fusion protein in PBS and 100 or 500 µg/ml BSA.

RESULTS

Isolation and characterization of tiggrin proteinThe culture media of Drosophila cell lines are useful sourcesof ECM proteins (Fessler and Fessler, 1989; Fessler et al.,1994). Tiggrin was isolated from the Drosophila Kc-derived167 cell line and antibodies were raised against it. SDS-PAGEanalysis of purified tiggrin indicates a single polypeptide,unchanged by reduction and therefore devoid of disulfidelinkages (Fig. 1, lanes 1, 2). The apparent Mr is approximately220×103, relative to a myosin marker and to several ECMproteins. Affinity purified antibodies to tiggrin react with it(Fig. 1, lanes 3, 4), but do not stain other Drosophila ECMproteins found in cell-conditioned culture media (Fig. 1, lane5). Some minor bands, apparent just below the major 220×103

Mr tiggrin band are derived from tiggrin. They react with anti-bodies affinity-purified against the major band (Fig. 1, lanes 3,4) and give similar CNBr digestion patterns (not shown). Asingle 220×103 band of tiggrin is also obtained by immuno-precipitation from the 24-hour culture media of either Kc 167cells, or of Drosophila primary cell cultures, incubated with[35S]methionine and [35S]cysteine (not shown). This verifiesthat tiggrin is synthesized by these cells, and not derived fromthe serum supplement of culture media. Detergent extracts ofembryos made under reducing conditions and resolved bySDS-PAGE containing either 4.5% or 10% acrylamide only

Fig. 1. SDS-PAGE and western blot of tiggrin. (A) Coomassie bluestained SDS 4.5% polyacrylamide gel with reduced (lane 1) andnonreduced (lane 2) purified tiggrin, which migrate with an apparentMr of approx. 220×103. (B) Western blot of non-reduced (lane 3) andreduced (lane 4) purified tiggrin developed with affinity-purifiedantibodies to tiggrin. Affinity-purified antibody did not cross reactwith other reduced Drosophila ECM proteins: laminin, collagen IV,glutactin and peroxidasin, which were present at high concentrationsin lane 5. Markers with approximate, estimated Mr are: reduced (a)laminin A, 430×103; (b) laminin B1, 215×103; (d) laminin B2,185×103; (c) collagen IV; (e) peroxidasin, 170×103; (f) glutactin(Fessler and Fessler, 1989) and (g) prestained myosin, 205×103

(BRL).

1750 F. J. Fogerty and others

Fig. 2. Deduced amino acid sequence of tiggrin. The 2186 amino acid sequence was derived from conceptual translation of the 6,558nucleotide cDNA sequence. Translation begins at nucleotide 150, the position of the first AUG start codon. The amino acid residue number isindicated to the right of each line. The signal peptide cleavage site is marked with an open triangle. The regions of the sequence that wereverified by amino acid sequencing of the protein are underlined. The single cysteine is enclosed in a shaded ellipse. The potential N-linkedglycosylation sites are indicated by brackets above the sequence. The repetitive segments are delineated with bent arrows and are numbered.The 81 amino acid spacer separating the 14 contiguous repeats from repeats 15 and 16 is marked with a dashed line. The RGD cell attachmentmotif is enclosed in a shaded box. The potential LRE cell adhesion sequences are marked with solid circles above the sequence.

1751Tiggrin, Drosophila integrin ligand

show the 220×103 Mr band with its related peptides on westernblots (Fig. 5B). This indicates that the tiggrin stained by anti-bodies in tissues corresponds to the isolated protein.

Molecular sieve chromatography, which may be greatlyinfluenced by molecular shape, gives a main peak of tiggrinthat elutes somewhat earlier than thyroglobulin. An additional,minor peak of Mr≈290×103, relative to globular calibration

proteins, corresponds to unfolded tiggrin chains (not shown).The hydrodynamic shape equivalent to tiggrin molecules is arod, 3 nm in diameter, and 70 nm long for a molecule con-sisting of a single chain or 180 nm long for a homodimer. Thenumber of chains in one tiggrin molecule is not known. Thecalculation is based on tiggrin’s sedimentation coefficient s20,W= 6.45 ± 0.2 S (see Methods).

200 400 600 800 1000 1200 1400 1600 1800 2000

GOR α-Helices

C RGD

GOR Turns

GOR β-Sheets

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fig. 3. Domain structure of tiggrin. Tiggrin is represented with three domains: the N-terminal domain that contains the sole cysteine, the centraldomain containing the repetitive segments (shaded boxes), and the C-terminal domain containing the RGD cell attachment motif. Thesecondary structure of tiggrin was predicted using the method of Garnier et al. (1978). The structure predicted for a region is shown below theline indicating amino acid number (α helices, β sheets, turns). The upper line indicates the numbers of the amino acid residues.

Fig. 4. Sequence alignment of the tiggrin repeats. The amino acid sequences of the 16 repeats marked in Fig. 2 are shown as aligned bythe multiple sequence alignment program, PileUp. Gaps (-) were added to optimize alignments. The consensus sequence (cons) indicatesamino acids that are conserved in that position in 8 or more repeats, and/or share this position with a related amino acid. Shaded verticalcolumns indicate conserved residue positions that may be characteristic of this repeat. Columns of predominantly hydrophobic (phob, ø),basic (pos, p) or acidic (neg, n) residues are indicated. Columns of predominantly hydrophilic (hydr, h) residues, that lack a conservedcharge, are also marked. The numerical order of the repeats is shown on the left and the sequence number of the residue that ends eachrepeat is given on the right.

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cDNA cloning and sequence analysis of tiggrinA cDNA expression library of Kc 167 cells was screened withanti-tiggrin antibodies and gave a set of 5 clones. This provided2 kb of the 3′ end of the tiggrin sequence. A 6,895 bp cDNAclone containing the complete protein coding region was thenisolated from the pNB40 12-24 hour Drosophila embryolibrary of Brown and Kafatos, (1988). The sequence of thesingle open reading frame codes for 2,186 amino acids. Asshown in Fig. 2 this contains the N-terminal 7 amino acids oftiggrin that had been determined from the protein itself, and 8amino acids from a CNBr fragment . The initiation Met isfollowed by a signal peptide of 18 amino acids. Downstreamof the termination codon is a poly(A)+ signal followed furtherdownstream by a poly(A)+ tail.

The cDNA of tiggrin predicts a novel protein with amolecular mass of 255×103 Mr. There are no hydrophobicstretches long enough to span a lipid bilayer. The PROSITEdata library was searched for potential post-translational mod-ification sites and revealed 12 potential N-linked glycosylationsites (Fig. 2, brackets). Some of these sites are glycosylated

because tiggrin’s electrophoretic mobility is increased slightlyfollowing treatment with PNGase F (New England BioLabs),an enzyme that removes N-linked oligosaccharides (data notshown). The adhesive recognition sequence RGD is found intiggrin near the C terminus (Fig. 2, shaded box). We searchedthe sequence for other described cell adhesion sequences(Yamada, 1991) and found two copies of a putative adhesiverecognition sequence, Leu-Arg-Glu (LRE) (Hunter et al.,1989) (Fig. 2, solid circles).

The predicted secondary structure of tiggrin suggests that theprotein can be divided into three major regions (Fig. 3). Acentral repetitive domain of approximately 1,250 amino acidsextends from residues 496 to 1,743 and is predicted to containprimarily α helical structure and very little β sheet structure.It is flanked by the N-terminal domain (residues 1-495) whichcontains the sole Cys (Fig. 2, shaded ellipse) and the C-terminal domain (residues 1,744-2,186) which contains theRGD cell attachment motif.

The central domain is composed of 16 contiguous repeats(Fig. 2, bent arrows), except for an 81 amino acid spacer (Fig.2, dashed line) between repeats 14 and 15. Fig. 4 shows amultiple alignment of the repeats, each 73-77 amino acids long,generated by the program PileUp. Repeat 5 is the closest to theconsensus sequence. Pairwise comparisons of individualrepeats to repeat 5 with the BESTFIT program show 25-42%amino acid identity and 55-61% similarity. To search for otherproteins that might contain this type of repeat, the program Pro-fileMake was used to generate a profile sequence from thealigned repeats. A search of two protein databases, SwissProt(release 24) and Prot 99 with the program ProfileSearch failedto find significant matches to this profile sequence. Thissuggests that the tiggrin repeat is a novel protein motif. It islikely to have a high content of α-helix, with only 4 of the 16repeats containing a proline residue that is incompatible withα-helix. While each tiggrin repeat contains 4-5 potential heptadrepeats of hydrophobic residues, such as occur in coiled-coilα-helical structures, these heptad repeats do not form a con-tiguous end-to-end sequence (Lupas et al., 1991).

The GenBank EMBL (release 76) and SwissProt (release 24)protein databases were searched for potential similarities withany part of the total tiggrin sequence. No overall homologieswere found by the FASTA and BLAST programs. Weaksequence similarities (<20% identity) were indicated betweentiggrin’s region of tandem repeats and several filamentousproteins that contain coiled-coil α-helices, e.g. myosin,spectrin and dystrophin.

Tiggrin transcripts and protein duringembryogenesisThe tiggrin gene maps to region 26D1-2 of chromosome 2 byin situ hybridization. Fig. 5 shows Northern blots of tiggrinmRNA and immunoblots of tiggrin protein in extracts ofembryos collected over successive 2 hour intervals. A single7.0 kb tiggrin transcript is first detected at 6-8 hours of devel-opment, increases to a peak level at 12-14 hours and thendeclines to a low level by the end of embryogenesis (Fig.5A,C). Subsequently the level of tiggrin mRNA increases to asecond peak during the late 2nd to early 3rd larval instar period(data not shown). In contrast, tiggrin protein first appears at 8-10 hours and its level then continues to increase throughoutembryogenesis, even after tiggrin mRNA levels have declined

F. J. Fogerty and others

Fig. 5. Developmental profile of tiggrin RNA and extractable tiggrinprotein. A shows a northern blot of total RNA (20 µg/lane) extractedfrom embryos of the indicated age intervals and hybridized with atiggrin antisense RNA probe. Panel B shows a western blot ofdetergent extracts (100 µg/lane) of embryos of corresponding ages,immunostained with anti-tiggrin antibodies and reacted with 125I-labeled protein A. The corrected optical density was summed overeach band of autoradiograms A and B, and normalized (NIH-ImageSoftware, Wayne Rasband). C shows the relative levels of tiggrinRNA and extractable tiggrin protein at successive embryo ageintervals (hours).

1753Tiggrin, Drosophila integrin ligand

(Fig. 5B,C). Tiggrin protein is found throughout the larvalstages with a peak level at 3rd instar. Soluble tiggrin wasdetected by western blot analysis in the hemolymph collectedfrom 3rd instar larvae. At pupation the tiggrin level declinesmarkedly and the levels of extractable tiggrin are very low inpupae and adult flies (not shown). The apparent discrepancy

between changing RNA levels and rate of protein increase seenin Fig. 5C is not confined to tiggrin. The cells that expresstiggrin also make laminin and collagen IV. The rates offormation of these proteins are also not tightly coupled to thecorresponding RNA levels, which vary independently of eachother over this developmental period (Kusche-Gullberg et al.,

Fig. 6. In situhybridization andimmunostaining of wholemount Drosophilaembryos. Micrographs ofwhole-mount embryos (A-H), with their anteriorends to the left, are shownat different levels of focus.In situ hybridization withcDNA coding for tiggrin:tiggrin transcripts arelocalized in hemocytes(he) (A,B) and in the fatbody (fb) (B).Immunostaining withantibodies to tiggrinshows tiggrinconcentrated at muscleapodemes (a) (D,E,G andH). Tiggrin protein alsoappears in hemocytes(C,F) and in basementmembranes, e.g.surrounding the gut (g)(D,F). The commissures(c) of the nerve cord alsostain for tiggrin (H).Control preimmune serumdid not stain embryos.Immunostained sectionsof adult jump muscleshow that tiggrin islocalized at the surface ofthe muscle, at the site of Zbands, as seen at twolevels of focus (I,J). Barindicates 100 µm (A-H),25 µm (I,J).

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1992). We conclude that, at least for hemocytes and fat bodycells, it is inappropriate to make the common, simplifyingassumption of equating developmental RNA patterns withquantitatively matching expression of the correspondingproteins.

To determine the spatial expression of tiggrin duringembryogenesis, cDNA probes were hybridized to whole-mount embryos. Transcripts are first detected in hemocytesduring germ band retraction (stage 12). Two hours later manyhybridizing hemocytes are distributed throughout the embryoand transcripts are also detected in the fat body (Fig. 6A,B).Correspondingly, tiggrin protein is seen in hemocytes and laterin the fat body, indicating that these two tissues are theprincipal sources of embryonic tiggrin. Tiggrin accumulatesgenerally in basement membranes, e.g. in those associated withthe gut musculature. Early deposits of tiggrin are found atsegmental furrows, at sites where muscle apodemes form (Fig.6D), and later it is concentrated at these muscle-epidermisattachment sites (Fig. 6E,G,H). Affinity purified antibodiesagainst the RGD-containing fusion protein and against tiggrin

give the same staining patterns. A definite staining of theventral nerve cord commissures is evident with affinity purifiedantibodies against tiggrin or the fusion protein (Figs 6H, 7E).

In the larval somatic musculature, tiggrin is most prominentat the muscle-epidermal attachment sites. It is also present inthe ECM surrounding the somatic muscles and in the basementmembrane underlying the epidermis (not shown). At the Z-bands of adult striated jump muscles, tiggrin occurs at themuscle surface (Fig. 6I,J), where αPS2βPS integrin is found.

The distribution of tiggrin protein was analyzed in theapproximately 25% of embryos of the balanced mysXG43 line,which show an overt myospheroid phenotype and cannotexpress the ßPS integrin chain (Mackrell et al., 1988; Leptin etal., 1989). Apparently normal muscle attachment sites initiallyform in myospheroid embryos, though muscles subsequentlydetach (Wright, 1960; Newman and Wright, 1981; Volk et al.,1990). There are other abberrations, the most prominent beingdorsal herniation. Antibodies to tiggrin, and against the RGD-containing fusion protein, clearly stain residual muscle inser-tions and react strongly with the dorsal herniation masses (Fig.

F. J. Fogerty and others

Fig. 7. Immunostaining of whole mounts of myospheroid embryos. Self crosses of the Drosophila (l) mysXG43 line gave rise to all the embryosshown in this figure. About 25% of the embryos of a cross have the myospheroid phenotype (A-C); the wild-type phenotype is shown in D-F.The control (D) was stained with preimmune serum. All other embryos were reacted with purified anti-tiggrin RGD-containing fusion proteinas first antibody. Apodemes (a) are stained both in the wild-type phenotype (E,F) and, in as far as they exist, in the myospheroid phenotype(A,B). Gut (g) and pharynx (ph) musculatures are also stained. Comparison with Fig. 6 illustrates that purified antibodies raised against tiggrinand against RGD-containing fusion protein give the same staining patterns, including the commissures (c) of the nerve cord in E. Bar indicates100 µm.

1755Tiggrin, Drosophila integrin ligand

7A-C). We tentatively conclude that ßPS integrins are notessential for the localization of tiggrin, though experiments arestill underway to exclude the potential effects in myospheroidembryos of small maternal contributions of myospheroid geneproducts (Wieschaus and Noell, 1986).

Individual hemocytes each synthesize several of the identi-fied ECM proteins: collagen IV, laminin, papilin and peroxi-dasin (unpublished data). To assess whether tiggrin is synthe-sized by these same hemocytes, whole-mount embryos wereimmunostained with mouse anti-tiggrin and rabbit anti-collagen IV antibodies. Of the hemocytes in 10 embryos a totalof 614 cells were clearly distinguished; 93-100% of these cellsin any embryo had reacted with both antibodies.

Previously we showed that primary Drosophila embryo cellsfrom postgastrulation embryos differentiated when cultured ona substratum of Drosophila laminin or human vitronectin(Volk et al., 1990; Gullberg et al., 1994). We have now testedas substrata tiggrin, and a fusion protein of tiggrin (residues1,891-2,161) that contains the RGD binding site. Excellentdifferentiation of several different cell types occurred; e.g.abundant multinucleate myotubes and neurites formed andboth hemocytes and clusters of epidermal cells were presentafter 18 hours of culture at 22˚C. On control coverslips coatedwith bovine serum albumin very limited differentiation wasobserved.

Tiggrin mediates cell spreading via αPS2βPSintegrinsThe finding of an RGD cell attachment motif in tiggrin, whichconspicuously colocates with both ßPS integrins at muscleapodemes and at Z-bands, emphasized the need to test tiggrinas an integrin ligand. To do this, we assayed the ability oftiggrin-coated substratum tomediate the spreading ofDrosophila S2 cells that have beentransformed with genes for αPS2and ßPS integrin chains (Bunch andBrower, 1992; Zavortink et al.,1993). A pair of alternative RNAsplice variants code for two formsof the αPS2 integrin chain thatdiffer by 25 amino acids near theputative ligand binding site(Brown et al., 1989). Correspond-ingly, two transformed S2 celllines were created: HSPS2(C),which expresses the αPS2 chainthat contains the additional 25amino acids, and HSPS2(m8) inwhich these amino acids (that arecoded for by exon 8) are missing.Both cell lines also express the βPSchain and make comparableamounts of integrins.

Tiggrin serves as a substratumfor αPS2βPS integrin-mediated cellspreading. HSPS2(C) cells showno spreading when applied to tissueculture plastic that has beenblocked with milk solids to preventnonspecific interactions (Fig. 8A).

When the plastic is first coated with tiggrin, these cells showextensive cell spreading (Fig. 8B). Similar but less extensivespreading is seen for HSPS2(m8) cells (see Fig. 9). The untrans-formed S2 cells show no spreading on tiggrin (Fig. 8C), demon-strating that the observed spreading is mediated by the integrinsexpressed from the transgenes. Spreading of both cell lines isabolished when the monoclonal antibody aBG-1, which blocksintegrin-mediated aggregation of Drosophila cells (Hirano etal., 1991), is added to the spreading medium (Fig. 9). Incontrast, the control CF.6G11 antibody (Brower et al., 1984),which binds to βPS integrin chains on cells, but does not blocktheir function, has no effect on cell spreading (Fig. 9). Tiggrinthat was eluted from an SDS-PAGE band also promotes celladhesion and spreading, providing further evidence that theactive cell adhesive component is tiggrin and not some chro-matographically unresolved contaminant. Furthermore, thefusion protein that contains 270 amino acids from the C-terminal region of tiggrin (residues 1,891-2,161), including theRGD sequence, but lacking both of the putative LRE cellattachment sequences and the central repeat domain, alsopromotes adhesion and spreading of >65% of HSPS2(m8) cellsand >85% of HSPS2(C) cells. The amounts of intact purifiedtiggrin or tiggrin fusion protein required to promote spreadingare similar. A control fusion protein, derived from the antisensestrand of the cDNA that codes for the RGD-containing fusionprotein, has no effect on cell attachment and spreading.

Cell spreading was quantitated on surfaces coated with 1µg/ml or 5 µg/ml tiggrin solutions (Fig. 9). Tiggrin mediatesthe spreading of a high percentage of cells expressing eitherαPS2βPS integrin. However, on plastic coated with 1 µg/mltiggrin the HSPS2(C) cells spread better than the HSPS2(m8)cells.

Fig. 8. HSPS2(C) Cell spreading on tiggrin. Shown is a phase contrast view of HSPS2(C) cellstransformed with genes that express αPS2(C)βPS integrin (A,B), driven by a heat shock promoter.These cells do not exhibit spreading on the uncoated substrate (A) but do spread on tiggrin (B).HSPS2(m8) cells also spread on tiggrin (not shown). Also shown are the untransformed DrosophilaS2 cells (C) plated on tiggrin; these cells do not spread on any substrate that we have tested. Theconcentration of tiggrin used to coat the plastic in this experiment was 5 µg/ml. Scale bar equals50 µm.

1756

To test the functional significance of tiggrin’s RGDsequence in these cell spreading experiments, we measured theeffects of increasing concentrations of an added, soluble RGDpeptide (GRGDSP) (Fig. 10). This peptide inhibits spreadingof both HSPS2(C) and HSPS2(m8) cells on tiggrin. Inhibitionof HSPS2(m8) cells is achieved with much lower levels ofsoluble peptide than is seen for the HSPS2(C) cells. This isprobably due to the lower initial levels of spreading seen forthe HSPS2(m8) cells in the absence of any peptide. The controlRGE peptide (GRGESP) has relatively little effect on cellspreading.

DISCUSSION

Tiggrin is a novel, RGD-containing Drosophila ECM proteinwhich is the first proven ligand of αPS2βPS integrins, animportant receptor group for the interactions of Drosophilacells with ECM. Tiggrin’s prominent association with muscles,especially at the muscle insertions, suggests primarily amechanical function, such as has been ascribed to the integrinswith which it interacts. The relatively late time of expressionof tiggrin during embryogenesis suggests that, like collagen IV,it may serve primarily to consolidate emerging organ struc-tures. While both proteins are made by the same hemocytes,and continue to accumulate in later embryos, the messagelevels of tiggrin fall while those of collagen IV increase(Kusche-Gullberg et al., 1992).

The elongated structure of tiggrin is consistent with itsproposed connector function between muscle and tendon cells,and with the shape of other ECM ligands of integrins such asfibronectin. The amino acid sequence of the central, majorportion of tiggrin is indicative of a high α helix content asfound in many filamentous proteins, but full biophysical inves-

tigations await isolation of sufficient quantities of protein. Thisregion of tiggrin comprises 16 copies of a well-conserved,novel repeat in which a distinct pattern of certain hydrophobic,polar and charged groups is maintained (Fig. 4). The majorityof amino acid residues, 72%, are charged or polar, and the ratioof charged to apolar residues of approximately 1.0, suggests anextended structure (Cohen and Parry, 1986). The structure pre-diction algorithms indicate interrupted α-helices, though thereare hardly any incompatabilities with α-helical folding. Themaximum total length of this central 1,250 residue domainfolded entirely as an α helix is 1,250×0.146 nm (helix rise perresidue)≈180 nm. To form a functional tensile entity there aretwo principal possibilities for stabilizing such an extended αhelix by coiled-coil interactions. (1) The α helices of two orthree separate tiggrin chains could mutually stabilize eachother, in analogy to the α-helical coiled-coils of myosin orlaminin. Correspondingly, each molecule would be ahomodimer, or homotrimer, of tiggrin chains, about 180 nmlong. Alternatively, (2), a single tiggrin polypeptide chain mayalso repeatedly fold back on itself to form a succession of α-helical, triple coiled-coil structures as in spectrin, α-actinin anddystrophin (Cohen and Parry, 1986; Conway and Parry, 1991).For a single tiggrin chain, in this folded-back type of coiled-coil structure, a maximal length somewhat less than180 nm/3=60 nm is predicted for the central domain. Theapproximate molecular lengths of the rod-like tiggrinmolecules that are predicted from tiggrin’s sedimentation coef-ficient are, respectively, 70 nm and 180 nm for molecules con-sisting of one or two chains. This includes the terminaldomains that are likely to be folded differently from the centralone (Fig. 3). Electron microscopy has been initiated todetermine the shape and dimensions of tiggrin molecules andto decide between the above possibilities.

The cell spreading experiments demonstrate that the inter-actions of αPS2βPS integrins with tiggrin depend critically onits RGD region. Although the adjacent Gln residue of

F. J. Fogerty and others

Fig. 9. Integrin-mediated cell spreading on tiggrin. HSPS2(m8) andHSPS2(C) cells were plated in plastic wells that had been coatedwith tiggrin at 0, 1, and 5 µg/ml and later the percentages of spreadcells were scored. Both cell lines show high levels of spreading onplastic coated with 5 or 1 µg/ml tiggrin, but not on uncoated plastic.This spreading was eliminated by the βPS function blockingantibody, aBG-1, but not by the control anti-βPS antibody, CF.6G11.As in Fig. 8, S2 cells showed no spreading on tiggrin.

Fig. 10. RGD inhibition of cell spreading on tiggrin. HSPS2(C) cells(triangles) and HSPS2(m8) cells (circles) were plated on plasticcoated with 1 µg/ml tiggrin. RGD peptide (GRGDSP; solid lines)shows a concentration dependent inhibition of cell spreading whilethe control RGE peptide (GRGESP; dashed lines) shows relativelylittle effect.

1757Tiggrin, Drosophila integrin ligand

..KDRGDQPPHT.. is unusual, a subsequent Pro residue alsooccurs in fibronectin. The peptide folding algorithms predict aturn at this part of the sequence, which may expose the ligandsite (see Main et al., 1992). An unusual cluster of Pro residuesfollows the RGD sequence. While a fusion protein encom-passing only the C-terminal domain serves as a ligand forαPS2βPS integrins, we do not know whether tiggrin has other,non-RGD, integrin binding sites and we have not investigatedthe potential function of the LRE regions.

The cell spreading experiments demonstrate that tiggrin cansupport αPS2CβPS- and αPS2m8βPS-mediated cell spreading. Ahigher percentage of the cells expressing the C form of αPS2, ascompared to those expressing the m8 form, spread on low con-centrations of native tiggrin (Fig. 9) or tiggrin fusion protein.That this represents a difference in the interactions between theseintegrins and tiggrin, and not just a difference in the levels ofintegrins expressed in the two cell lines, is supported by theobservation that cells expressing αPS2m8βPS integrin show morespreading on vertebrate fibronenctin than those expressingαPS2CβPS integrin (Zavortink et al., 1993; This finding wasrepeated in experiments done in parallel with the tiggrin cellspreading experiments). These results suggest that the develop-mental changes in the splicing of mRNA encoding the αPS2chain (Brown et al., 1989) may provide a mechanism for mod-ulating the binding of tiggrin to the αPS2βPS integrins in vivo.

For tiggrin to function as a connector linking cells to eachother or to matrix, each tiggrin molecule is likely to bind toseveral components. The rich ECMs of both Drosophila andvertebrate muscle insertions suggest that multiple, partlyredundant interactions between numerous components assurephysiologically reliable function. Characteristically, thegenetic absence of a Drosophila integrin does not block thedevelopmental assembly of somatic muscles but only weakensthem (Wright, 1960; Zusman et al., 1990, 1993; Brabant andBrower, 1993). In keeping with this, we suggest that the ECMcomponents in the initially formed myospheroid apodeme areadequate for deposition and attachment of tiggrin, and that thebinding of tiggrin to integrin and ECM components contributesto the strength of normal muscle attachments. The finding oftiggrin within the ventral nerve cord also suggests that, inaddition to integrins, tiggrin binds to other molecules.

We thank Drs H. P. Bachinger, B. Blumberg, R. Doolittle, D.Eisenberg, D. Godt and D. Rice for help and advice, and one of thereviewers for encouraging us to include our findings on myospheroidmutants. We appreciate reagents and instruments made available tous by Drs N. Brown, S. Crews, J. L. Couderc, D. Dean, K. Garrison,R. Levine, Y. Sun and M. Takeichi. We thank Dr A. Fowler forprotein sequencing. Protein sequencing performed at the UCLAProtein Microsequencing Facility was aided by a BRS Shared Instru-mentation Grant 1 S10RR05554-01 from the N.I.H. This research wassupported by grants from the Muscular Dystrophy Association,National Institutes of Health AG02128 and the UCLA AcademicSenate to JHF, and NIH grant GM42474 to DLB, and NationalResearch Service Awards 5-T-32-CA09056-16 and 5-F32-HD07476-02 to F. J. F.

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(Accepted 24 March 1994)

F. J. Fogerty and others

Note added in proofThe Genbank Accession Number for the nucleic acid sequenceof tiggrin is U 09506.