A 33 kDa protein with sequence homology to the 'laminin binding protein'

is associated with the cytoskeleton in hydra and in mammalian cells

E. KEPPEL and H. C. SCHALLER

Center for Molecular Biology (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg 1, FRG

Summary

In hydra and in mammalian cells the monoclonalantibody V recognises an epitope which colocaliseswith cytoskeletal structures. Using this antibody forexpression screening, a cDNA clone (955 bp) wasisolated from hydra, which covers an open readingframe for a protein of 294 amino acids with acalculated molecular mass of 32.8 kDa. Northern blotanalysis of hydra RNA resulted in a single mRNAspecies of 1.2 kb, and primer extension experimentsproved this to be the full length message. 218 residuesat the amino terminus of the hydra protein showextensive homology (73.5%) to a human proteindesignated 'laminin binding protein'. The carboxyl-terminal 76 amino acids possess no significantsimilarity (20%). The monoclonal antibody V, whichrecognises an epitope in this carboxyl-terminal part,reacts in Western blots, both in hydra and in

mammalian cells, with a protein of 33 kDa and notwith the 45 kDa 'laminin binding protein'. The 33 kDaprotein is not extracellular or transmembrane, buthas a strictly intracellular location as indicated by itsamino acid sequence and by immunocytochemicaland cell fractionation studies.

In non-dividing mammalian cells the 33 kDa pro-tein colocalises with filamentous structures; in div-iding cells it dissociates from it and concentratescentrally. Presence of the SPLR-sequence, which isthe consensus phosphorylation motif for the p34cdc2

kinase, links this 33 kDa protein to events occurringduring the cell cycle.

Key words: hydra, cytoskeleton, expression screening, human'laminin binding protein', p34cdc2 phosphorylation motif, celldivision.

Introduction

In the freshwater coelenterate hydra, cellular growth iscontrolled by the nervous system. At appropriate timesnerve cells release neuropeptides like head and footactivator which act on surrounding cells as growth factors(Schaller et al. 19896). The effect of head activator on celldivision is visible both in hydra and in mammalian cellsl h after application as an increase of cells in mitosis(Schaller et al. 1989a). This indicates that within this shortperiod of time signal transduction occurs via interaction ofthe peptide with a membrane receptor, followed by signalamplification over second messenger systems, leading toreorganisation of the cytoskeleton, condensation ofchromosomes, nuclear envelope breakdown, formation ofthe mitotic apparatus, segregation of sister chromosomesand finally, cell division.

In an attempt to understand this pathway we investi-gated changes in cytoskeletal organisation preceding celldivision. In this paper we describe a 33 kDa protein withits full length cDNA clone. This protein is associated withfilamentous cytoskeletal elements in hydra and in mam-malian cells. Like other cytoskeletal structures it under-goes reorganisation during mitosis. The possibility thatphosphorylation of the 33 kDa protein might regulate thisreorganisation is supported by the presence of theconsensus phosphorylation motif for the cell cycle-specificp34cdc2 kinase.Journal of Cell Science 100, 789-797 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

Materials and methods

AnimalsHydra vulgaris (formerly named Hydra attenuate) was originallyobtained from P. Tardent, Zurich. The multiheaded mutant of thespecies Chlorohydra viridissima was from H. M. Lenhoff, Irvine.The animals were cultured in a medium consisting of 1 mM CaCl2,0.1 mM MgCl2, 0.1 mM KC1 buffered with 0.5 mM sodiumphosphate, pH7.6, (hydra medium), at 19(+2)°C. Animals werefed daily between 9 and 10 a.m. and washed 5-7 h later. Forexperiments they were starved for at least one day.

Preparation of hydra cell fractionsHydra were collected from the culture, sedimented and hom-ogenised in a buffer containing 0.4 M sucrose in hydra medium towhich 1 mM phenylmethylsulfonyl fluoride (PMSF) was added asprotease inhibitor (buffer A). All steps were carried out at 4°C. Onaverage, 1000 Hydra vulgaris yielded a volume of 1 ml containing10 mg of protein. After the first gentle homogenisation the tissuewas centrifuged at 1000 # for lOmin. The sediment wasredissolved in buffer A, rehomogenised and centrifuged again at1000 g for 10 min to yield the nuclear and cytoskeletal pellet. Thecombined supernatants were centrifuged at 50 000 g for 30 min toyield the membrane pellet. This 50 000 # sediment was redis-solved in buffer A without sucrose and recentrifuged at 50 000 gfor 30 min.

AntibodiesThe IgM monoclonal antibody V was originally produced andselected against head activator-containing conjugates (Schawal-

789

ler et al. 1988). Hybridoma cell supernatants were concentrated10- to 20-fold by (NH^SC^ precipitation and used at dilutions of1:100 to 1:5000.

Actin detectionFor colocalisation studies with actin, the rhodamine-coupledphytotoxin phalloidin (a kind gift from Michael Nassal) was usedat a concentration of 0.5 ̂ g ml"1 (Wieland, 1981; Jahn, 1982).

DNA stainingCells were incubated for lOmin at room temperature with

r 1 Hoechst 33258.

ImmunocytochemistryHydra. Whole animals of the species Hydra vulgans were fixed

overnight in Lavdowsky's fixative, a solution containing 4%acetic acid, 3.7% formaldehyde and 50% ethanol (Dunne et al.1985), or alternatively in 4% paraformaldehyde in phosphate-buffered saline (PBS: 140 mM sodium chloride, 10 mM sodiumphosphate, pH7.2). Lavdowsky's fixative was better for visual-ising filaments with either an a-actin antibody or with themonoclonal antibody V, whereas formaldehyde was better forstaining with phalloidin. The animals were then washed threetimes for 30min in PBS and then once with PBS, 0.1% TritonX-100. Nonspecific binding was blocked by incubation for 30 minat room temperature in PBS with 1% bovine serum albumin(BSA) and 0.1% Triton X-100. The monoclonal antibody V wasused at a dilution of 1:100 in the same solution and incubatedovernight at 4°C. After washing three times for 15 min in PBS,0.1% Triton X-100 and blocking for 30 min, animals wereincubated for 3-4 h at room temperature with fluoresceinisothiocyanate (FITC)-labelled o--mouse IgM at a dilution of 1:100in PBS, 0.1% Triton X-100, 1% BSA. The preparations werewashed twice in PBS for 10 min and then counterstained withrhodamine-labelled phalloidin (0.5 fig ml"1) for l h at roomtemperature.

NIH3T3 mouse fibroblasts. The procedures described above forwhole mounts of hydra were also used for the study of NIH 3T3mouse fibroblasts grown on tissue culture slides.

Immunoblotting of proteinsTotal hydra protein samples were prepared by boiling polyps inSDS sample buffer (62.5 mM Tris/HCl, pH6.8, 2% SDS, 5% /S-mercaptoethanol, 5% glycerol, 0.01% bromophenol blue). Cellfractions were either dissolved directly in sample buffer or afterconcentration by ethanol precipitation. Proteins were fraction-ated by electrophoresis on an SDS-polyacrylamide gel (Laemmli,1970) and transferred onto nitrocellulose filters (Towbin et al.1979). Blots were saturated for 30 min in blocking buffercontaining PBS, 10% fetal calf serum (FCS), 1% BSA, 0.2%casein and then incubated overnight at 4°C with the firstantibody, V, diluted 1:5000 in the same buffer. After three washeswith PBS containing 0.05% Triton X-100, the second antibody(Sigma, rabbit cr-mouse IgM, alkaline phosphatase-coupled) wasapplied at a dilution of 1:1000 in blocking buffer and theincubation was continued for 3h at room temperature. Thenitrocellulose was then washed again three times with PBS,0.05 % Triton X-100, then once in PBS without Triton X-100, andfinally with a buffer containing 100 mM Tris/HCl, 100 mM sodiumchloride, 5mM MgCl2, pH9.5 (AP buffer). The alkaline phospha-tase-linked second antibody was then detected via a colourreaction by incubation of the nitrocellulose in AP buffercontaining 0.33 g I"1 p-nitro blue tetrazolium chloride, 0.17 g I"1

5-bromo-4-chloro-3-indolyl phosphate. The enzyme reaction wasstopped with 20 mM Tris/HCl, pH 8, 5 mM EDTA.

Molecular biologyTwo cDNA libraries of the multiheaded mutant of the speciesChlorohydra viridissima were constructed following the methodof Huynh et al. (1985), using the expression vector Agtll. TheAgtll library was screened with the V antibody at a dilution of1:5000. Antibody binding was detected using an alkalinephosphatase- labelled rabbit cv-mouse IgM (Sigma, 1:1000).

To obtain the full length clone a cDNA insert, isolated via theAgtll screening, was radiolabelled with 32P and used for furtherscreening of an oligo-dT-primed AgtlO library of the multiheadedmutant Chlorohydra viridissima, constructed as described inBenton and Davis (1977). For sequence determination, cDNAinserts were cloned into the plasmid vector pBS. DNA sequencingwas done using the chain termination method (Sanger et al. 1977).DNA and amino acid data were analysed using the HUSARsoftware package (Heidelberg Unix Sequence Analysis Re-sources).

Southern and Northern blot analysisGenomic DNA was digested with the restriction endonucleases.EcoRI and ffindlll, fractionated by agarose gel electrophoresisand transferred to nitrocellulose filters. Hybridisation to filter-bound DNA (Southern, 1975) was carried out in a solutioncontaining 43% formamide, 5xSSC (150 mM sodium chloride,15 mM sodium citrate), 50 mM sodium phosphate, pH7.0, 0.1%sodium dodecyl sulfate (SDS), 200 fig ml"1 tRNA, lOxDenhardt's(0.02% BSA, 0.02% Ficoll, 0.02% polyvinyl-pyrrolidon) at 42°Covernight. After hybridisation, filters were washed with 2xSSC,0.1 % SDS at room temperature and twice with 1 xSSC, 0.1 % SDSat 42°C. For Northern blot analysis, 5^g poly(A)+ RNA waselectrophoresed through a 1 % agarose gel in 3.7 % formaldehyde,20 mM morpholine propane sulfonic acid, 50 mM sodium acetateand 10 mM EDTA, pH7.0. The RNA was transferred to a HybondN membrane (Amersham) and hybridised with the radiolabelledcDNA clone (Thomas, 1980) in a solution containing 50%formamide, 5mM sodium phosphate, pH6.7, 5xSSC, 0.1% SDS,lmM EDTA, lxDenhardt's, 200/jgmr1 salmon sperm DNA.After an overnight incubation at 50 °C the membrane was washedwith lxSSC, 1 % SDS. The DNA fragments used as hybridisationprobes were labelled with 32P by using the multirandom primemethod (Feinberg and Vogelstein, 1983).

Results

Immunocytochemical colocalisation of the V antigen withcytoskeletal structures in hydraThe freshwater coelenterate hydra consists of two celllayers, ectoderm and endoderm, which contain epithelio-muscular cells as main constituents. Such cells have anapico-basal polarity with muscular processes located attheir base (Kanaev, 1952). In these processes, actin-containing fibers are oriented parallel to the body axis inthe ectoderm and perpendicular to it in the endoderm(Lentz, 1966). Fig. 1 shows these fibres in whole polypsstained with rhodamine-coupled phalloidin, a phytotoxinwhich binds filamentous actin (Wieland, 1981). Themonoclonal antibody V also recognises these actin-containing fibers, shown as a double stain in Fig. 2. Inaddition to this filamentous appearance of the V-antigen,we also detected a diffuse cytoplasmic staining of dividinginterstitial cells in hydra.

Identification of a 33 kDa protein as a target for themonoclonal antibody V in hydraTo establish the identity of the protein containing theepitope for the monoclonal antibody V, Western blotanalyses of extracts from whole hydra were performed.One major area of immunoreactivity was detected as adoublet band with a molecular mass around 33 kDa(Fig. 3). The extract derived from one hydra sufficed todetect this band, indicating that the 33 kDa protein isrelatively abundant (0.1-1% of all proteins).

Cloning and characterisation of the cDNA coding for the33 kDa protein from hydraThe monoclonal antibody V was used for immunoscreen-

790 E. Keppel and H. C. Schaller

Fig. 1. Visualisation of actin filaments in whole mounts of Hydra vulgaris. Animals were fixed with 4 % paraformaldehyde andstained with rhodamine-coupled phalloidin. (A) Young bud still attached to the mother animal. (B) Tentacles surrounding ahypostome with longitudinal fibres in the ectoderm and horizontal fibres in the endoderm around the mouth opening. (C) Headregion with two tentacle bases. (D) Part of a tentacle in which both longitudinal and horizontal fibres are visible. Bars, A, 100 /an;B, 35 /rnij C, 30 /an; D, 25/an,

Fig. 2. Colocalisation of the V antigen with actin filaments. Extended tentacles from Hydra vulgaris were stained with rhodamine-coupled phalloidin to visualise actin (A) and with the V antibody which was made visible by a FITC-coupled second antibody (B).For fixation, 4 % paraformaldehyde was used. Bar, 20 /an.

Sequence of a cytoskeletal protein in hydra 791

1 2 3 4KDa

- 6 6

- 4 5

- 3 6

CGGGTGAGTTTATCCTGATAAGGTAACCATAATGTCTGAGGGAATTGATGCTCTTTCTCTH S E G I D A L S L 10

- 2 9

-24

20

Fig. 3. Western blot analysis ofextracts from whole hydra of thespecies Hydra uulgaris with themonoclonal antibody V. Lane 1was a negative control with thesecond antibody alone. Lanes 2-4were incubated with themonoclonal antibody V at adilution of 1:5000. Lanes 1, 2, 3and 4 contained 50 fig, 10 fig, 30 ngand 100 fig of protein, respectively.Molecular mass standards aredepicted by bars.

ing of an oligo-dT-primed Agtll library from Chlorohydraviridissima. One out of 10 recombinants gave positivesignals. Five clones were sequenced and all possessed thesame 3' end. The shortest clone detectable with the Vantibody coded for 150 bases. 45 of them belonged to the 3'untranslated region. From this it is concluded that the 35carboxyl-terminal amino acids, coded by the remaining105 bases, must contain the V epitope. The longest insertof 282 bp was used as a probe to screen an oligo-dT-primedAgtlO cDNA library of Chlorohydra viridissima for longerinserts. One clone with 955 bp contained the whole codingregion (Fig. 4). The first in frame ATG was found atposition 32-34. This start triplet was preceded by themotif CCA at position 27-29, which conformed to theconsensus translation initiation sequence (Kozak, 1984).This putative initiation codon was followed by an openreading frame of 882 bases ending at position 911 with theTAG termination codon. The coding region was followed by14-18 bases of a 3'untranslated sequence containing twopossible polyadenylation signals, AATAAA, (Proudfootand Brownlee, 1976), starting from position 928 and 932,respectively. The amino acid sequence predicted from thisopen reading frame was 294 residues long with acalculated molecular mass of 32.8 kDa and a theoreticalisoelectric point of 5.16.

Further computer analysis revealed the followingfeatures. The protein sequence contained no glycosylationsites, but several putative serine/threonine phosphoryl-ation sites. Four of these, located at positions 78, 190, 221and 286, could be signals for protein kinase C; one atresidue 252 could be a signal for casein kinase II, and onelocated at position 152 contained the motif SPLR, whichcorresponds to the consensus phosphorylation site for thep34cdci protein kinase (S/T-P-X-R) (Peter etal. 1990). Verystriking was the relatively high abundance and the

61 TAAAGAAGAAGATGTTGTCAAGrTCTTGGCAGCTGGAGTACATTTAGGTTCAACTAATGTK E E D V V K F L A A G V H L G S T N V 30

121 TGGATCATCTTGCCAAGGCTATGTTTTT AAAAGAAAAAGTGATGGTATTCACATCATT AAG S S C Q G Y V F K R K S D G I H I I N 50

181 CTTAAGAAAGACTTGGGAAAAACTTATTCTAGCAGCAAGAATTATTGCAAGCATTGAAAAL R K T K E K L I L A A R I I A S I E N 70

t

241 TCCTGCTGATGTTTGTGTTATATCTTCACGACCTTATGGAACACGTGCTGTTCTTAAATTP A D V C V I S S R P Y G T R A V L K F 90

301 TGCTCAATCAACAGGTGCCATACCTATTGCTGGTAGGTTTACTCCAGGAACATTTACAAAA Q S T G A I P I A G R F T P G T F T N 110

361 TCAAATTCAAAAACGATTCCGTGAGCCTCGCTTGTTAATTTCTACTGATCCACAGCATGAQ I Q K R F R E P R L L I S T D P Q H D 130

421 TAATCAGGCATTAACTGAAGCTTCATATGTAAACATTCCTGTTATTGCCTTATGTAACACN Q A L T E A S Y V N I P V I A L C N T 150

4 81 GGATTCGCCTTTGAGATTTGTTGACTGTGCTATTCCATGTAATAATAGAGGTATTCAATCD S P L R F V D C A I P C N N R G I Q S 170

541 AATTGGTACTATGTGGTGGATATTAGCAAGAGAGGTATTGCACCTCCGTGGAATAATCAGI G T M W N I L A R E V L H L R G I I S 190

601 TCGCAAGACACCATGGAATGTCATGCCTGATTTATTCTTTTACCGTGATCCTGAAGATATR K T P W N V M P D L F F Y R D P E D I 210

661 TGAAAAGGAAGAGCAGGCTGCAGCTATTGCCTCTGCTAAACCTGATGAACCTTATCAGCCE K E E Q A A A I A S A K P D E P Y Q P 230

721 TGATTTCTCTGGAAATGTTGATCAATCTGCTGCAGGCGCTGACTGGGGAGATCAACCAGTD F S G N V D Q S A A G A D W G D Q P V 250

781 TGTTACTGGTGCTGATTGGACAGCTGAACCAAGTGTATCAAAGGATTGGGCTGCTGAACCV T G A D W T A E P S V S K D W A A E P 270

841 TGCTGGCTGGGAGGCTGATACAACTGCTGTGTCTGGTGATTGGGCAACCCCTAAGACAGAA G W E A D T T A V S G D H A T P K T E 290

901 AGACTGGGCTTAGTTTAACTGTTTTAAAATAAATAAAAAAGTTGCTCTTGAAAAAD W A

Fig. 4. Nucleotide sequence and predicted amino acid sequenceof the cDNA clone encoding the 33 kDa protein. The nucleotidesequence was determined by the dideoxy method (Sanger et al.1977). Numbering of bases starts at the first nucleotide, that ofamino acids at the first methionine as potential start site ofthe ORF. Thin lines indicate the position of start and stopcodons and of the two possible polyadenylation signals. Thickunderlining indicates the potential phosphorylation site of thep34cdc2 kinase. Stars indicate possible serine/threoninephosphorylation sites. Arrowheads label the tryptophanresidues. The open circle indicates a putative myristoylationsite.

pattern of the usually rare amino acid tryptophane: 10tryptophane residues were present in the 294 amino acidsequence, 6 of them, preceded by aspartic acid in 5 out of 6cases, were located with a relatively even spacing of 6-10residues in the last 50 amino acids at the carboxylterminus of the molecule. The results of hydropathyanalysis, performed according to the method of Kyte andDoolittle (1982) confirmed the hypothesis that this is ahydrophilic protein containing some weak hydrophobicregions (Fig. 5A). Hydropathy analysis performed accord-ing to the method of Engelman et al. (1986) (Fig. 5B)revealed that there was no stretch of amino acids long andhydrophobic enough to serve as a transmembrane region.A putative myristoylation site at amino acid position 4, aglycine residue, followed by a serine at position 5, mayindicate membrane anchoring and/or protein-proteininteraction (Mcllhinney, 1990).

So far two genes have been cloned from hydra, actin(Fisher and Bode, 1989) and src-related protein (Bosch etal. 1989). Both have been shown to be very A+T-rich, actincontaining 75% and src-related protein 82% A+T. The882 coding bases for the 33 kDa protein consist of 250 A,198 G, 164 C and 270 T, corresponding to a total A+Tcontent of 59%. This is not distributed randomly, but

792 E. Keppel and H. C. Schaller

120 150 ISO 2i0

Sequence number

preferentially (75%) located at the third position of acodon, indicating a preference for A or T at the wobbleposition for the hydra codon usage.

Northern blot analysis of poly(A)+RNA from hydra, inwhich the radiolabelled cDNA of the 33 kDa protein wasused as a probe, revealed the presence of a single RNAspecies of about 1.2 kb (Fig. 6). Primer extension exper-iments showed this to be the full length mRNA. No largermRNA species were detected.

To characterise the genomic organisation of the geneencoding the 33 kDa protein, Southern blot analysis wasperformed using genomic DNA of hydra, digested eitherwith the restriction enzyme EcoRl or with HindUl, andhybridised with the radiolabelled 955bp-long cDNAencoding the 33 kDa protein. The ScoRI-digested DNA(Fig. 7, lane 1) showed one single band in the range of6000 bp. The HindUl digest resulted in two bands withsizes of 4000 and 1400 bp, respectively (Fig. 7 lane 2). Thisis in agreement with an internal Hmdlll site within thesequence encoding the 33 kDa protein. From this weconclude that the gene encoding the 33 kDa protein occursonly once in the hydra genome.

Sequence homology of the hydra 33 kDa protein with a'laminin binding protein'A computer-directed homology search revealed a strikinghomology between the first 218 amino acids of the 33 kDahydra protein and a human 'laminin binding protein'(Wewer et al. 1986; Yow et al. 1988), amounting to 73.5%homology at the amino acid level and to 65% at thenucleotide level (Fig. 8). The carboxyl-terminal part (76

— 74

-4 .4

-2.4

kb

-1.4

-0,24

Fig. 5. Hydropathy analysis ofthe putative amino acid sequenceof the 33 kDa protein. Datapresented as hydrophilic portionsare plotted above the horizontalline, hydrophobic regions areshown below the horizontal line.(A) Hydropathy analysisaccording to Kyte and Doolittle(1982). (B) Hydropathy analysisaccording to Engelman et al.(1986).

Fig. 6. Northern blot analysis ofpoly(A)+ RNA isolated from themultiheaded mutantChlorohydra viridissima. RNA(5 fig) was electrophoresed,blotted and hybridised with theradiolabelled cDNA encoding the33 kDa protein.

amino acids), which in the human protein is supposed to beresponsible for laminin binding (Wewer et al. 1986), andwhich in hydra contains the V epitope, did not show anysequence homology.

Sequence of a cytoskeletal protein in hydra 793

kb 1 2 3 4 5 6 7 8 9 1 0

ft- 6 , 3- 4 . 3

-1 ,4Fig. 7. Southern blot analysisof genomic DNA from themultiheaded mutantChlorohydra viridissima.Genomic DNA digested withthe restriction ezyme EcoBI(lane 1) or Hindfn. (lane 2) wasseparated on an agarose gel,blotted and hybridised with theradiolabelled cDNA cloneencoding the 33 kDa protein.

Is the 33 kDa protein a laminin binding protein?To establish a possible relationship between the humanand hydra proteins the V antibody was assayed in Westernblots with extracts from human cell lines. In contrast tothe putative 'laminin binding protein' which was shown tomigrate to a position of 40-45 kDa (Van den Ouweland etal. 1989; Rabacchi et al. 1990) the V antibody reacted inhuman as well as in hydra with a 33-35 kDa protein (datanot shown).

tmmunocytochemistry revealed no binding of the Vantibody to the cell surface on living cells. This indicatedthat the carboxyl part of the 33 kDa protein which wasrecognised by the V antibody and which, in the case of thehuman 'laminin binding protein' was predicted to bindlaminin, was not outside the cell. To prove the intracellu-lar localisation of the V antigen, cell fractionation studieswere performed. Analyses of the different hydra cellfractions showed that under mild iso-osmolar homogenis-ation conditions about 90 % of the 33 kDa protein cosedi-mented with nuclear and cytoskeletal components at

hydra

human

hydra

human

hydra

human

hydra

human

hydra

human

hydra

human

MSEGIDALSLKUDWKTLAAGVHLGSTHVSSSCQGYVFKRKSDGIHIINII I I II I I I I I I II I I I I II I I I I I I II I I I

MSSlOOVLQKKUDVLXrLAAGTHLGGTNLDrQMEQyiYKRKSDGIYIIN 50

LMTWEXLILAMIIASIENPADVCVISSRPYSTRAVLKFAQSTGAIPIA 1 0 0I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ILKHTWKLLLAARAIVAIIKPXDVSVISSRMTGORAVLKTAAATGATPIA 1 0 0

GRTTPGTTTMQIOKJirBIPRLLISTDPQHDNQALTEASrVNIPVIALCNT 1 5 0I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

SRrrPGTTTHQIOAAfREPiaLWTDPRADHQPLTtASYVNLPTIALCNT ISO

DSPLRPVDCAIPCNNHGIQSIGTVWWILARIVLHLRGIISRKTPHNVMPD 2 0 0I I I I I I I I I I M l I I I I I I I I I I I I I I I I I I I I I I IDSPLRYVDIAIPCNNKGAHSVGLHiniMLAREVUtMRGTISREHPMEVMPD 200

LFFYRDPEDIEKEEOAMIASAKPDEPYQPDFSGNVDQSAAG ADWG 246I I I I I I I I I I I I N I I I I I I I I ILYFYRDPEEIEKEEQAAA. EKAVTKEEFQGEItTAPAPEFTATQPEVADllS 2 4 9

DQPWTGADKTAZPSVSKDKAAEPAGWEADTTAVSGDXATPKTEDHA-I I I I I I I I

EGVQVFSVFIQQFPTEDNSAQPATEDNSAAFTAQATEHVGATTDHS*

293

- 6 6

—45

— 36

- 2 9

- 2 4

Fig. 9. Western blot analysis of cell fractions from Hydravulgaris with the monoclonal antibody V. Cell fractionationwas performed as described in Materials and methods. Lanes1-3 contained the 1000 g sediment with nuclear andcytoskeletal components, lanes 4-6 contained the 1000 gsupernatant with the soluble proteins, lanes 7-9 contained the50 000 # sediment with the membrane components. Lane 10contained the 50 000 g supernatant. Different amounts of hydraequivalents were separated on a 12.5 % SDS-polyacrylamidegel, as follows: 3 (lane 1), 5 (lane 2), 10 (lanes 3, 4, 7), 30(lanes 5 and 8), 50 (lanes 6, 9 and 10).

1000 g, 5-10% cosedimented with the membrane fractionat 50 000 g, and 0-5% were present in the 50 000 gsupernatant in a soluble form (Fig. 9). By extracting the1000 g sediment with salt (80 HIM NaCl), or 0.6 M KI,which is supposed to depolymerise filamentous actin(Jahn, 1982) and other cytoskeletal components, or byhomogenising cells at acidic pH (10 mM ammoniumacetate, pH 6) the 33 kDa protein was converted quantitat-ively into the soluble form. This suggests that the 33 kDaprotein is an intracellular component, most of which is

Fig. 8. Comparison of the deduced amino acid sequence of thehydra 33 kDa protein (upper line) with the human 'lamininbinding protein' (lower line).

Fig. 10. Association of the V antigen with filamentousstructures in NIH 3T3 mouse fibroblasts. Cells were fixed withLavdowsky's fixative. Bar, 15 /an.

794 E. Keppel and H. C. Schaller

11A

Fig. 11. Change in the V epitope localisation during cell division in mouse fibroblasts. NIH 3T3 cells were fixed with Lawdowsky'sfixative. Shown are phase contrast (A, D, G, J) and fluorescence micrographs of cells stained either with the V antibody inconjunction with a FITC-labeled second antibody (B, E, H, K) or with Hoechst to visualise the DNA (C, F, I, L). Different mitoticstages are indicated by arrowheads. Bar, 35 /an.

Sequence of a cytoskeletal protein in hydra 795

attached in vivo to cytoskeletal structures. This issupported by immunocytochemistry on fixed NIH 3T3mouse fibroblasts, where the V epitope is associated withcytoskeletal structures (Fig. 10). In interphase cells the Vantibody reacted with thin filaments extending over thewhole cell (Fig. 10 and Fig. 11). Staining of the filamen-tous structures disappeared when the cells started to entermitosis, as shown in Fig. 11 where different mitotic stageswere visualized by staining the cells with Hoechst(arrowheads). There was an accumulating immunoreac-tivity around the nucleus when chromosome condensationoccurred. As mitosis proceeded through metaphase andanaphase, the V antigen appeared as a bright, diffusecytoplasmic staining (Fig. 11 D-I). When cytokinesis wasalmost complete filamentous structure became visibleagain (Fig. 11 J-L). These observations indicate that thechange in localisation of the 33 kDa protein is correlatedwith changes occuring during the cell cycle.

Discussion

Eucaryotic cells have a high degree of internal organisa-tion brought about by a complex network of proteinfilaments which are responsible for distinct cell shapes.Different parts of the cytoskeleton must be linked togetherand their functions must be coordinated in order tomediate intracellular events. During mitosis the cytoskel-etal network undergoes a series of dramatic structuralchanges, which finally leads to cell division.

Several lines of evidence presented in this study haveallowed us to conclude that a 33 kDa protein, which isrecognised by the monoclonal antibody V, is involved incytoskeletal rearrangements during mitosis. In wholemounts of hydra, this antibody reacted on the one handwith actin-containing filaments in muscular processes ofepithelial cells, and on the other hand appeared as adiffuse cytoplasmic pattern in dividing interstitial cells.Similar results were obtained with mammalian cells,where in differentiated NIH 3T3 fibroblasts, for example,the V epitope was associated with cytoskeletal structurescontaining actin filaments, but in dividing cells itdissociated from the filaments and concentrated centrally.The immunocytochemical colocalisation with intracellu-lar filamentous structures was confirmed by cell fraction-ation studies, which showed a copurification of the Vimmunoreactivity with cytoskeletal elements.

To characterise the immunoreactive 33 kDa antigen atthe nucleotide level, we used the V antibody for expressionscreening of a Agtll cDNA library from hydra. The fulllength cDNA clone obtained encoded 294 amino acids.Primer extension experiments confirmed the fact that theclone contained the whole coding region for a protein witha calculated molecular mass of 32.8 kDa. This corre-sponded to the V immunoreactive protein which inWestern blot analysis migrated with 33 kDa.

Comparison of the putative amino acid sequence withother known proteins revealed a striking homologybetween this hydra protein and a human protein desig-nated 'laminin binding protein' (Yow et al. 1988). At theamino acid level, this homology amounted to 73.5 %, at thenucleotide level to 65 %. The homology between these twoproteins was restricted to the 218 amino-terminal aminoacids. The remaining 76 amino acids showed no significanthomology. This part of the human protein was originallydescribed as the laminin binding domain of a 67 kDa'laminin receptor' (Wewer et al. 1986).

For the human and the hydra protein only a singlemRNA species of 1.2 kb was detected and no largertranscripts. 67 kDa would exceed this coding capacity.Since the encoded proteins have similar general features,including lack of a signal peptide and an internalhydrophobic stretch long enough for transmembranespanning, we consider it unlikely that they are com-ponents of the outer cell membrane. We confirmed this bydemonstrating that living cells do not expose the proteinat their outer cell surface and that the 33 kDa proteincopurified with cytoskeletal and not with membranefractions.

The hydra and the human protein contained severalconserved serines and threonines as putative phosphoryl-ation sites for kinases, which was in accordance with theappearence of the 33 kDa as a doublet band in Westernblots. Increased levels of phosphorylation, for example ofvimentin or laminin, brought about by interaction withthe p34cdc2 kinase during mitosis, are accompanied byfilament disassembly and nuclear envelope breakdown(Ottaviano and Gerace, 1985; Miake-Lye and Kirschner,1985; Dessev and Goldman, 1988; Chou et al. 1990).Especially interesting for the hydra protein in this contextwas the presence of the sequence SPLR, which correspondsto the consensus phosphorylation motif for this cell cycle-specific kinase. We suggest that phosphorylation of thissite may trigger dissociation of the 33 kDa protein fromactin-containing filaments, thus initiating its reorganis-ation during mitosis.

We wish to thank Imke Scheurlen and Graeme Bilbe forconstructing the cDNA libraries. Phalloidin was a kind gift fromMichael Nassal. We thank Sabine Hoffmeister for critical readingof the manuscript and Silke Druffel-Augustin for excellenttechnical assistance. This work was supported by the DeutscheForschungsgemeinschaft (SFB 317), by the Bundesministeriumfur Forschung und Technologie (BCT 365/1) and by the Fonds derDeutschen Chemischen Industrie.

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(Received 29 April 1991 - Accepted, in revised form, 4 September 1991)

Sequence of a cytoskeletal protein in hydra 797