A novel extensin that may organize extracellular matrix biogenesis in Volvox carteri

A novel extensin that ^may organize extracellular matrix biogenesis in Volvox carteri

Hans Ertl, Armin Hallmann, Stephan Wenzl and Manfred Sumper

LehrstuhlBiochemie I, Universitat Regensburg, 8400 Regensburg, FRG

Communicated by J.Schell

ISGis a sulphated,extracellularglycoprotein synthesized for onlyafew minutesininverting Volvox embryos and invertingsperm cellpackets. This control operates at the level of transcription. ISG has been characterized by studies of protein chemistryandelectronmicroscopy. The primary structure of ISG has been derived from genomic DNA andcDNA. ISG is composed of a globular and a rod-shapeddomain. The rod-shaped domain represents amemberof theextensinfamilywithnumerous repeats of Ser-

(Hyp)4.6

motifs. A synthetic decapeptide matching theC-terminalsequenceis able to disaggregate theorganisminto individualcells. Immunofluorescence microscopy localizes ISG withinthe boundary zone of the ECM.

Key words: extensin/extracellular matrix/ISG/Volvox

Introduction

Members of the genus Volvox are among the simplest multicellularorganisms. They are composed of only two cell types: somatic andreproductivecells. Thereproductivecycle of Volvox carterihas beendescribed in detail(Starr, 1969, 1970). The asexual organism exhibits 2000-4000 biflagellatedsomaticcells(similartoChlamydomonasintheir morphology) thatarearranged in a monolayer at the surface of a hollowsphere.Sixteen muchlarger asexual reproductive cells ('gonidia') liejust below the somatic cells. The cells are held together by a complex, glycoprotein-rich extracellular matrix (ECM). Eleven or twelve rapid and synchronous cleavage divisions of agonidium generate all the cellsof the adult organism. The resulting embryonic cells arealready arrangedinahollowsphere,buttheir orientation with respect to the surface is the reverse of that found in theadult: theflagellarendsof the somatic cellsaredirected towards the centre of thesphereandgonidiaprotrudefrom the surface. Duringinversion, theembryo turns completely inside outthroughacross-shapedslit(the'phialopore')and thereby establishes the adultconfiguration (Pickett-Heaps, 1970;

Kelland,

1977; ViamontesandKirk, 1977; Viamontes etal., 1979; Greenetal., 1981).Inversion isinitiated when cellsborderingthephialopore undergoatransition in

shape

and begin to resemble the classic 'bottle cells' of the gastrulating amphibian embryo

(Holtfreter, 1943, 1944).

Next, the sequence of cell shape changes progresses in a

wave-like fashiontowards thepoleoppositethe

phialopore.

After inversion, the somatic cells

begin

to secrete ECM

material

causing

eachcelltomoveapart from its

neighbours.

The organism now grows in size but not cell number.

The organizationofthe Volvox ECM has been

analysed

atthe

light-

andelectron-microscopiclevels. The details of ECMarchitecture have been summarized and a system of nomenclatureproposedbyKirketal. (1986).Each

organism

is surrounded by a boundary zone which includes a crystalline lattice that is very similar to that of Chlamydomonasreinhardtii(GoodenoughandHeuser,

1988;

Adair and Appel, 1989; for review, see Roberts et

al.,

1985). The ECM internal to the boundary zone has been designated cellularzone. Themostdistinct structuralelement ofthis zone isahighly regularpattern of fibrous layers that surround cells at a distance to form contiguous cellular compartments. A sulphated, hydroxyproline-rich glycoprotein (SSG 185)hasrecently beencharacterized as the monomeric precursor of this ECM substructure

(Ertl

etal., 1989).

Themechanismbywhich Volvox cells manage theprecise construction of all these defined structures atconsiderable distance from their own boundaries is unknown. In this paper, we characterize theinversion-specific glycoprotein (ISG) that is synthesized for only a few minutes during embryonicinversion and we present evidence for afunctional role of ISG in the organization ofECM architecture.

Results

ISG was previously discovered by pulse labelling experimentswith radioactive sulphate and characterizedas asulphatedglycoprotein (apparentmolecularmass200kDa) that is synthesized exclusively in inverting embryos for a period of < 10min(Schlipfenbacheretal., 1986). Although ISG issynthesizedonly duringthis extremely shortperiod in the 48 h life cycle of Volvox, the mature glycoprotein remains stable for at least 24h, as revealedby apulse-chase experiment with radioactive sulphate (Figure lA).

A previously described purification procedure (Schlipfenbacher et al., 1986) apparently yields homo- geneous material asjudged bySDS-polyacrylamide (5%) gel electrophoresis. However, higher percentage polyacrylamide gels revealed that this material was still contaminatedby aglycoprotein,sothepurificationprocedure was modified as follows.

Purification of ISG

Aunique feature of sexual development in the male strain which leads to spheroids containing sperm cells is the occurrence of an additional morphogenetic process: in the first inversion process theembryoturnsinsideout asduring asexual development. Twentyhours later eachreproductive cell (now called an androgonidium) undergoes a series of cleavagestoform apacketof sperm cells. This bowl-shaped sperm cell mass again performs a morphogenetic process

A B A ^{2 3} B ^{1 2}

...

.....:

s a ..:

:.

F) ^{1 . .}

Fig. 1. Pulse labeling of ISG with [35S]sulphate. (A) Volvoxspheroids containing embryos at the inversion stage werepulse labelled for 30 min(lane 1) followed bya20hchase(lane2). Fluorograms of SDS-polyacrylamide gels (6%) loaded with the crude

ECM/membrane fractionfrom the Volvox lysates. R, authentic ISG.

(B)Asingle male spheroidatthe desired stage ofdevelopmentwas selected under thestereomicroscope, pulse labelled for 10 min with [35S]sulphate andlysed in SDS samplebuffer. The lysatewasapplied to anSDS-polyacrylamide gel(6%). The individual spheroids contained uncleaved androgonidia (lane 1), androgonidiaatthe initiation ofspermatogenesis (lane 2), sperm packetsatthe initiation of inversion (lane 3), inverting sperm cell packets (lane 4), spermpackets atthe end of inversion(lane 5) and sperm packets 60 min after the end ofinversion (lane 6). R, authentic ISG. For detailsseeMaterials andmethods.

(secondinversion)toformaspermbundlewhichisconvex on its anterior side (Starr, 1969). ISG

synthesis

is

again

initiated during this second inversion. The period of ISG synthesis during spermatogenesis was

analysed by pulse

labelling of single Volvox spheroids (to circumvent the problem ofasynchronous developmentin the

population).

As shown in Figure

iB,

ISG

production during

spermatogenesis is

again

restricted to the short

period

of inversion.

Atthe firstinversionthe ratioof somatic cells and

embryos

is ^- 2000:16, whereas the ratio of somatic cells and sperm cellpackets (atthe second

inversion)

is ^- 1:1.This

explains

thehighercontentofISG in

spheroids containing

spermcell packets which therefore served as the source for ISG purification.

ISG wasextractedfrom the spermpackets

by incubating

thespheroids in 0.8 MNaCl for12 hat4°C. After removal ofthe spheroids by centrifugation, ISG in the extract was adsorbedto ananion

exchange

resin

(Q-Sepharose).

After extensivewashing, thehighly sulphatedISGcould

only

be elutedatNaCl concentrations ashighas 1.35 M.

Although

ISG represents a minor component of the salt extract, it becomeshighly enriched after thisstepandonlyafew other sulphated glycoproteins contaminate the ISG preparation (Figure 2A,lane 1). Finalpurificationtohomogeneity was achievedby preparative SDS-PAGE (lane 2). ISGisonly partially digested by proteinaseK: remarkably, a resistant corematerial remains whichexhibitsanevenhigherapparent molecular mass on SDS -PAGE(Figure 2A, lane 3).This behaviouriseasily explainedonthe basis of the ISGprimary structure (see below).

Treatment of ISG with anhydrous hydrogen fluoride at 0 °C,aprocedure that selectively cleaves glycosidic bonds (Mortand Lamport, 1977) drastically reduces the apparent molecular mass to -60 kDa (Figure 2B). This result indicates a high degree of glycosylation. By quantitative

Fig.2. Purification anddegradation of ISG.(A) Crude ISG preparation after anionexchangechromatographyonQ-Sepharose (lane 1), the final ISGpreparation(lane 2) and ISG afterdigestion withproteinase K (lane 3). TheSDS-polyacrylamide gel (6%)was stainedwith Stains All. (B)ISGbefore(lane 1) and afterchemical deglycosylationwithhydrogenfluoride(lane 2).A silver stain of the SDS-polyacrylamide gel (8%) is shown.

sugardeterminations(Duboiset

al., 1956),

the

carbohydrate

content wasdetermined to be 70%.

Protein chemistry studies

Automated Edman degradation of the purified ISG glycoproteinshowedthat its N-terminal amino acidsequence was AVSYSVSVYNNIAVTGAPLSGIVSQLLSK. The purified ISGglycoproteinwasdigestedwithtrypsin andthe resulting peptide mixturewasseparatedby reversed phase HPLC. Thematerials of wellseparated peaksweredirectly subjectedtoamino acid sequenceanalysisonanautomated gasphasesequencer.The aminoacidsequencedataobtained aresummarized inTable I.After chemicaldeglycosylation of ISG using anhydrous hydrogen fluoride, subsequent digestionwithtrypsin generateda setofadditional peptides.

A remarkable feature of these peptides is their extremely high content ofhydroxyproline residues (Table I).

Generation ofa 363 bp cDNA probe by PCR

The amino acid sequence of the N-terminus was used to synthesizeasenseoligonucleotideprimer, GTNTAT/CAA T/CAAT/C AT T/C/A GC, corresponding to amino acids 8-13. Theantisenseprimer, GG G/T AGGTTNCCGAA G/ATT,wasdesignedfrom amino acid positions 6-11 of peptide4 (Table I). The latter primer was used to reverse transcribe mRNAisolated from inverting sperm packets. The resulting cDNA was amplified by the polymerase chain reaction usingboth the sense and antisense primers. Thirty cyclesofamplificationproduced a DNA fragment of -360 bp in length that was cloned by blunt-end ligation into pUC18. Sequencing of this fragment revealed an open reading frame matching the amino acid sequences of peptides

Table I. Aminoacid sequences of tryptic peptides derived from ISG Peptide Amino acid sequence

I Val Asn Asp Leu Leu Thr Phe Val Arg

2 Gly Gln Gly Ser Tyr TrpIleThrGluGly Leu ThrSer - Ser ThrLys

3 AsnGly Gly Ser LeuIle Leu Val Asn Gly Ala Asn Gly Asn Asp Asn Thr PheIle Pro Leu 4 Ile Asp Pro Pro Ser Asn PheGlyAsn Leu ProVal Lys

Obtainedonly fromdeglycosylated ISG:

5 SerHyp HypHyp Hyp Hyp Arg Val Hyp Hyp Ser Hyp Hyp Hyp HypVal Ala Ser Hyp Hyp Hyp Hyp Hyp Hyp Hyp Arg 6 AlaSer Hyp Hyp Hyp HypHyp Ala Ser Ser Hyp Hyp Hyp Hyp Hyp Arg Hyp Hyp Hyp Hyp Ser Hyp Hyp Hyp Ser Hyp Hyp Hyp

HypAla ThrAla Ala Ala Asn Hyp Hyp Ser Hyp Ala Hyp Ser Arg

7 SerSerHyp Ser HypHyp Hyp Hyp Val Val Ser Hyp Hyp Hyp Hyp Hyp Hyp Arg

1-4 (Table I). Therefore this DNAfragmentwasused as a highly specific probe for the cloning of the ISG gene.

Cloning of the ISG gene

From previous experience with the cloning of a hydroxyproline-rich glycoproteinfrom Volvox (Ertl etal., 1989) itappearedunlikelythat a fulllength cDNAcovering theextreme cytidine-rich regionencoding the polyproline stretch wouldbedetected. Indeed, repeatedscreening of a Xgtl1 cDNAlibraryusingthe PCR probe mentioned above wasunsuccessful. Tocircumvent these problems, the PCR probe was used to screen a genomic library of V.carteri constructed in the replacement vector XEMBL 3. Out of 50 000 phages screened, three positive clones were identified. Digestionofthe 16kb insert of one of these clones with BamHI resulted in a 2.7 kb fragment that hybridized to the 363 bp probe. This fragment was subcloned into pUC18 andsequenced. AsshownschematicallyinFigure 3, the 2.7 kbfragmentcoveredthe completecoding region of the ISG gene. Theaminoacidsequence isencodedin three exons. Exon 1encodesatypicalsignalsequenceinaddition toaminoacidpositions1-4of the maturepolypeptidechain.

Exon 2 encodes amino acids 5-207 and exon 3 encodes an extremely long stretch ofprolines and hydroxy amino acids. Alloftheprolinesin this stretchanalysed bypeptide sequencingwerefoundtobehydroxyprolines (Table

1).

The deduced cDNA and amino acid sequence is given in Figure4. The correctassignmentof exon-intron boundaries was confirmed by sequencing the corresponding cDNA fragments. These cDNAs were generated by reverse transcription of mRNA using synthetic antisense oligonucleotidesandsubsequentamplification byPCR with ISG-specificsenseandantisenseoligonucleotides. Figure 3 schematically summarizes the strategy

applied

in orderto collectthe complete cDNA sequence information.

Primarystructure ofISG

TheN- andC-terminal halves ofthe

polypeptide

chain have completelydifferent amino acidcompositions. AnN-terminal stretch of 207 amino acids is

composed

of all

naturally

occurringaminoacids.Insharpcontrast, theC-terminalhalf (248 amino acids) of the polypeptide consists almost exclusivelyof

hydroxyprolines,

serines andthreonineswith arginineresiduesinterspersedwithin the clusters of

hydroxy

amino acids. Motifs of

Ser-(Hyp)3-7

are a

predominant

feature. Theserepeatsare

diagnostic

of

extensins,

a

family

ofrod-like,

hydroxyproline-rich glycoproteins

found in the

Genomic DNA

I =

E c

mI

£D

Q c

IZ E

(V (V

W'

I

cDNA

100 bp

Fig. 3. Restriction maps of the ISG gene and of cDNA fragments generatedby reverse transcription of mRNA followed by PCR amplificationtoconfirmputativeexon-intron boundaries. Black bars represent exons.

cellwallsofhigher plants (reviewed by Cassab and Vamer, 1988).

ISG is highly glycosylated as indicated by the drastic reduction of the apparent molecular mass after chemical deglycosylation with anhydrous hydrogen fluoride.

Arabinose, galactose and xylose in a ratio of 2:1:0.2 were showntobe the main sugarconstituents of ISG (Wenzl and Sumper, 1982). The deduced amino acid sequence exhibits a single N-glycosylation site at Asn74 that is indeed glycosylated as indicated by the amino acid sequencing of the corresponding peptide (Table I). Since the hydroxyproline-rich C-terminal domain of ISG is sensitive todigestionwithtrypsinorproteinaseKonly after treatment with anhydrous hydrogen fluoride (Table I), glycosylation of allor mostof the hydroxy aminoacids is highly likely.

Electron microscopy

Purified ISG and protease derivedfragments thereofwere studied by electron microscopy applying the rotary shadowingtechnique. IntactISG iscomposedofa

globular

domain andarod-likestructure. Themeanlengthof the rod was determined to be 57 i 3 nm. Both monomers and oligomericaggregatesaredetectable;the latter appearasstar- like particles with a variable number of the 57 nm arms

(Figure 5). Theproportions of different

oligomers

were as follows: 37% ofparticles had two arms, 18% had three arms, 12% fourarms, 10% five arms, 4% six arms, 4%

seven arms and 14% more than seven arms. Protease treatment convertsISG intoarod-like molecule of average length56 4 nm.Most

probably,

the

secondary

structure of the

hydroxyproline-rich

partof the

polypeptide

chain is the

polyproline

II helix conformation. This is the most

-

1 GCT GTT TCA TAT TCT GTA AGC GTC TAC AAC AAC ATC GCG GTC ACA GGG GCT CCC CTC TCT GGC

1 A V S Y S V S V Y N N I A V T G A P L S G

64 ATC GTG TCT CAG TTG CTA TCC AAA TGG AAG CTC AAT GTT CCC ACT TTG AGG ACA GTC TAC TCC

22 I V S Q L L S K W K L N V P T L R T V Y S

127 CAG CCG AGC GCT GCA GAG TTG TCA AGC ACC AAC GCC TTT ATC GTA TAC TCC AAG GGT CAG GGC

43 Q P S A A E L S S T N A F I V Y S K G Q G

190 TCC TAC TGG ATT ACG GAA GGC CTG ACC TCG AAC TCA ACT AAG GTT AAC GAT CTA CTC ACA TTT

64 S Y W I T E G L T S N S T K V N D L L T F

253 GTC CGT AAT GGA GGT TCC CTT ATC CTT GTC AAC GGC GCC AAC GGA AAT GAC AAC ACA TTT ATT

85 V R N G G S L I L V N G A N G N D N T F I

316 CCT CTT ATT CAC GCG CTG ACT GGC GGG GAT ACT CTC TGC ATC GCG AGG AGC TAC GCA GAT GAC

106 P L I H A L T G G D T L C I A R S Y A D D

379 ACT CGC ATC TAC CGT CGC ATC GAC CCT CCA TCC AAC TTT GGC AAC CTG CCT GTC AAG CAG TTC

127 T R I Y R R I D P P S N F G N L P V K Q F

442 CGC TAC ACT GCG GAT CTG TAT ATT ACC GGC CTA GAC TGC TTA TCT GGC ACC TCT ATT TAT TCC

148 R Y T A D L Y I T G L D C L S G T S I Y S

505 169 568 190 631 211 694 232

TCC GAC CCA ACC AAA AAG CTT TAC GCC ATC TCT GCC GGC ATC ACA TGG AGC GTG GGA CAG GGC

S D P T K K L Y A I S A G I T W S V G Q G

GCC GTG ACG TGG GTC GGC GCC GAC ATT GTG GCT GAC TCC AAG AAC ACC GTA GCC TTG GTG ACA

A V T W V G A D I V A D S K N T V A L V T

GCT GCG GCG GTC GTC GTA CAG ACA ACC CCG TCG CCG CCG CCG CCG CCA CGA GTT TCA ACG TCG

A A A V V V Q T T P IS P P P P AR V S T rS

CCG CCG CCA CCA GCC CGT GTC TCA TCC TCG CCG CCG CCC GCC ACG CGC TCG CCG CCA CCC CGT

P P P P | A R V S S |S P Pp A T R Is P P P R

757 CGT ATA ACG TCT CCT TCA CCA GTC CTC ACT GCA TCC CCA CCA CTC CCG AAA ACA TCG CCA CCA

253 R I T S P S P V L T A S P P L P K T |S P p

820 CCG CCG CCG CGC GTC CCG CCC TCG CCG CCA CCA CCG GTT GCT TCT CCG CCG CCA CCA CCA CCT

274 p p P R V P p S P P P P V A IS P P P P P P

883 CCA CGC GTC TCC CCG TCG CCG CCT CCG CCG CAG CCA GTT TCC TCT CCT CCC CCA CCC CCG CCA

295 7 R V S P IS P P p P-| Q P V S s P p p p p p

946 CCG CGC CCT TCA CCC TCG CCG CCG CCT CCA CGT TCT TCA CCC TCG CCG CC¢ CCG CCA AGT CCT

316 - R P S P S p p p P R S S P sS P P P P

1009 CCT CCT CCT TCT CCC CCG CCA CCG CGC CCT TCA CCC TCG CCO CCG CCT CCA CGT TCT TCA CCC

337 p p P S P P P P R P S p S P P P

PI

^{R S S P}

1072 358 1135 379 1198 400 1261 421 1324 442 1387 463

TCG CCT CCA CCG CCA GTC GTT TCG CCT CCG CCG CCG CCA CCC CGC GCC TCC CCA CCA CA CCT

S P P P P V V r P P P P P p R A IS P P P p

CCG GCA TCC TCT CCG CCG CCG CCG CCT CGC CCT CCT CCT CCC TCG CCT CCT CCC TCG CCT CCG

IJA S S P P P p p R P P p p I P pP p IS P p

CCG CCN GCT ACC GCT GCC GCC AAC CCT CCA TCT CCT GCC CCC AGC CGC AGC CGG GCC GGT GGG

P p A T A A A N P P S P A P S R S R A G G

CCC CCC CTT GGC ACA CGT CCT CCT CCT CCC CCT CcO GAG GAT GAT GCA CCG CCG CCG GAC TAC

P P L G T R P P P P P P Z D D A P P P D Y

TAC TTC CCC CCG CCC CAG GAC ATG TCG CCO CCO CCA CCC AAG AAG AAG GCA ACT GOC CGO CGO

Y F P P P Q D M s P P P P K K K A T G R R

CTC CTC TAA

L L *

Fig. 4. Nucleotide sequence ofcDNA andpredicted amino acid sequence ofmatureISG. Amino acid sequences derived fromisolated peptides are indicatedby broken lines. Ser-(Hyp)4-7 repeatswithin the C-terminal half of ISGareboxed. Thenucleotide sequencereported here has been submitted totheEMBLdatalibrary and is available under the accession number X65165 V.carteri mRNA ISG.

extended helix formedby polypeptideswith three residues Transcriptionalcontrol of ISG synthesis

perturn andapitch of0.94 nm. A rod, 57 nmin length, PCR was used to determine whether the extreme correspondsto apolypeptidechain of 180residues, a value developmental control of ISG synthesis operates at the level that is in good agreement with the length of the of transcription. RNA was extracted from a single embryo hydroxyproline-rich domain. collected at a defined stage of inversion. Reverse

-. ~~~~~~~~~. j -~~~~~~~~~~~~~ ~ ~ ~ ~~'*~~~~~j

^%

Fig. 5. Visualization of ISG by electron microscopy after rotary shadowing. Selected star-like particles witha variable number of 57 + 3 nm long arms are shown. Ina randomly selected field, the number ofarmns on50 particles were evaluated (see text). Thebar corresponds to 100nm.

transcription and subsequent PCR amplification of ISG cDNA yielded the results shown in Figure 6. mRNA for ISGis virtuallyabsent atthe very beginning of the inversion process. However, a strong signal is obtained from inverting embryos. Towards the end ofinversion, the level of ISG mRNA decreases significantly. Thus, the developmental control of ISG production operates at the level of transcription.

3

0

4

N.../

In vivo effects ofa synthetic ISG-derived peptide A distinct feature at the C-terminal end of ISG is the occurrence offivenegatively charged amino acid residues betweenpositions433 and448 whichare followed byfive positively charged amino acid residues between positions455 and 464. A synthetic decapeptide (KKKATGRRLL) matching the latter amino acid sequence was added to developing embryosandanalysed for

possible

interference with ECMbiogenesis. Concentrationsaslowas5x

10-6

M disturb ECM biogenesis and cause the development of

irregularly

shaped spheroids. Ataconcentration of2x

10-5

Mthespheroid beginstodisintegrate,producing singlecells or cell clumps never observed in normal development (Figure 7). However, theviabilityof the cells isnotaffected since furtherdevelopmentof the

reproductive

cellsproceeds normally. Interestingly, thispeptide nolongerdisturbs the

organization

of the ECM ifit is added a few hours after embryonic inversion. Commercially available

peptides

containingpositively chargedaminoacids(GK, LRRASLG andKKGE)usedas acontrol didnotaffect ECMbiogenesis at all, even at concentrations of 10-4 M.

Indirectimmunofluorescence microscopy

The localization of ISG within the ECM was studied by indirect immunofluorescence microscopy

using

the

IgG

fraction ofa rabbit

polyclonal

antibody raised

against

the globular domain of ISG produced in Escherichia coli

by

recombinant DNA

technology.

For that purpose, intact Volvox spheroids were broken up into hemispheres and

304 bp -

Fig. 6.Transcriptional control ofISG synthesis. RNA was extracted fromasingle embryoatthe desired stageof inversion(embryos were selectedunder a stereo microscope). After reversetranscriptionwithan ISG-specific antisense oligonucleotide,a304 bp cDNAfragment of ISGwasamplified by PCR. Thesense and antisense oligonucleotides wereselected fromexons 1 and 2 respectively to prevent amplification ofgenomic DNA. RNAextractionwas done ateach of the inversion stages schematically illustrated above the corresponding lanes ofthe agarose gel (1.5%). Fordetails, seeMaterials and methods.

smallerfragments (to enable the antibodytopenetrate into theECM),simply by

forcing

them

through

adrawn

pipette.

The fragments were stained with anti-ISG

(purified IgG

t

2

2

Fig. 7. In vivoeffectsofasynthetic decapeptide (KKKATGRRLL) matchingthe C-terminal sequenceof ISG. Preinversion embryoswere

incubatedin Volvoxmediumcontaining l0-4M(A2)or2^x10-5 M (B2) peptide. Al and Bl show untreated controls. Photographs were

taken 24 h(A) or48 h (B)aftertheaddition ofpeptide.

fraction) and fluorescein-conjugated anti-rabbit IgG. The antibody selectively stains a narrow layer within the boundary zoneof theECMaswellastheflagellartunnels ofeach somatic cell (Figure 8C andD). The stainedlayer and the flagellar tunnels are contiguous. This feature is diagnostic ofboundary zone 2 of the ECM (according to the nomenclature introduced by Kirk etal., 1986). In inverted^spermcellpackets, theflagellar tunnels againare

strongly stained as are the cell-cell contacts between individual sperm cells (Figure 8E).

Discussion

The polypeptide chain of ISG consists oftwo completely different domains. The C-terminal half of thepolypeptide chain is composed of a many repeating units of (Ser -Hyp46) and has a rod-like structure. These characteristics arediagnostic ofa member ofthe extensin family. Extensins have been describedatprotein andgene levels from dicotyledonous plants (Cassab and Varner, 1988); they^are structural proteinsof the higher plant cell walls. Uponsecretiontothe cellwall,extensinsarerapidly insolubilized, presumably throughthe formation of covalent cross-links. Theyarethoughttocontributetothe structural integrity of the cell wall throughout plant development.

Furthermore, synthesis ofextensins is induced by fungal infection andwounding(Showalteretal., 1985). Although the ECMs ofalgaeintheorder Volvocalesaremade entirely from hydroxyproline-rich glycoproteins, most of the glycoproteins characterized so far were reported to be different from typical higher plant extensins (Adair and Appel, 1989; Ertletal., 1989). Incontrast,azygote-specific cell wallglycoprotein (Woessner and Goodenough, 1989) andISGarethefirsthydroxyproline-richglycoproteins from

algae shown to be closely related to the extensins fromhigher plants.

The extensin domain of ISG is N-terminally extended by

>200 amino acid residues. This extension exhibits no unusual amino acid preferences and is visualized byelectron microscopy as a globular domain attached to a rod-like element. This additional protein domain supports the idea of a functional role of ISG. The extreme developmental control of ISG synthesis further supports this idea. ISG is synthesized in embryos for only a few minutes towards the endofinversionandis alsosynthesizedin inverting sperm packets. Duringinversion, all cellsoftheembryo are joined by numerous cytoplasmic bridges (Green et al., 1981).

Immediately after inversion these bridges disappear in V. carteri and the production of ECM material is initiated.

Itisprobablyanimportant function of one of the early ECM glycoproteinstohold the embryonic cells together, thereby replacing the function ofthe cytoplasmic bridges. ISG is probablythefirst ECM component produced in the embryo and, therefore, ISG is a candidate forbeing involved in this function. The in vivo effects caused by an ISG-derived peptide further support this idea. Synthesis of the glycoproteinSSG185,precursorof the insolubleECMsub- zoneCZ3(Figure 8A)is initiatedaslateas4-6 hafter the end ofinversion (S.Wenzl and M.Sumper, unpublished).

Unfortunately, ISG antibodiesdo not penetrateintactVolvox spheroids, preventingthe observation of effects caused under in vivo conditions.

The immunofluorescence data locate ISG within the boundaryzoneofECM,mostprobablywithin sub-zone BZ 2(Figure 8A)accordingtoKirk etal.(1986). Thissub-zone represents the tripartite layer, corresponding to layers W2-W6ofChlamydomonasin theterminology ofRoberts (1974)andincludesthechaotrope-solublecrystallinelayer.

The tripartite layer is highly conserved within the order Volvocales(Cattetal., 1978)and has been showntopossess the

ability

of

self-assembly

aswell as nucleatedassembly (Adairetal., 1987).Theobservationsdescribedinthispaper suggestthat ISG islikelytobe involved in theearlyprocesses ofECM biogenesis.

A unique feature of ISG synthesis is its extreme developmental control. Eventheexquisitely sensitive method of PCR amplification does not detect any ISG mRNA molecules until theinitiationofinversion.Thus, the control ofISGproduction is regulated atthe transcriptional level.

During the period between the first and second inversion events (sexual maledevelopment) only verylow levels of ISG mRNA aredetectableby PCRamplification, indicating the

instability

of thismessage. Inversion takes

place

atthe end of embryogenesis or spermatogenesis when all embryonic cells are differentiated. Since the ISG gene is activated in asexual embryosas wellas in spermpackets, thissignalcannotdependonaparticularly differentiated cell type. Acommonfeatureof cellsengagedintheprocessof inversionis achange of cell shape.Itis temptingtospeculate that thereorganizationofthe cytoskeletondeliversthesignal for ISG gene activation.

Materials and methods

Cultureconditions

Volvox carterif nagariensis, strains HK 10 (female) and 69-lb (male), wereobtained from the culture collection of algae at the University of Texas (Dr R.C.Starr). Synchronous cultures were grown in Volvox medium

A ¹

A

C

2

D

E

Fig.8. Localization by indirectimmunofluorescence of ISG in asexual spheroids (C and D) and in sperm cell packets (E). (A) Stylized drawing of a portionofa Volvoxcarterispheroid illustrating ECM zones and the corresponding nomenclature as proposed by Kirk et al. ( 1986). BZ,boundary zone;CZ, cellular zone; DZ, deepzone. (B)Westernblotanalysis of polyclonal antibody (purified IgG fraction) raised against recombinant ISG polypeptide (aminoacids5-172,see Materials andmethods). Lane 1, authentic ISG, lane 2, deglycosylated ISG. (C) Left, a somatic cell sheet photographedwith Nomarskioptics; right, immunofluorescence of thesamearea (stained withpurifiedantiISGIgG and FITC-labelled anti-rabbit IgG). Inthisorientation, bothflagellar tunnels of each somatic cell are mainly visible. (D) As (C), but a side view of somatic cells bordering the edgeof the sheet. (E) Left, inverted sperm packetsphotographed with Nomarski optics; right, immunofluorescence of the same area

(ProvasoliandPintner, 1959)at28°Cinan8 h dark/16 hlight(10000 lux) cycle(StarrandJaenicke, 1974).Large scalegrowthof strain 69-lb wasperformedaccordingto aprotocol describedpreviously (Tschochner etal., 1987).

Pulselabelling of ISG

Pulse labelingwith [35S]sulphate ^wasperformed asdescribedbyWenzl and Sumper (1986b). Pulse labelling ofa single Volvox spheroid was

performedas described by Schlipfenbacher etal. (1986) forindividual embryos.

Purification of ISG

Volvoxspheroids from six 20 1 cultures containing inverted sperm packets were harvestedby filtration throughanylonscreen.Thespheroidswere broken upbyforcingthemthrougha0.6mmhypodermicneedle. The cell suspensionwasadjustedto0.82MNaCI and incubatedovernightat4°C.

Aftercentrifugationat26 000gfor 20min, thesupernatantwasadjusted to1% N-lauroylsarcosine, 20mMTris-HCI(pH8.0)andheatedto95°C for 10min. Aftercoolingto4°C, Q-Sepharose correspondingto abed volumeof 10 mlwasadded andthesuspensionwasstirredovernight.The suspensionwaspouredintoacolumn and theQ-Sepharosewassubsequently

washed with 100 ml ofasolutioncontaining8Murea,0.85MNaCI and 0.5% N-lauroylsarcosine. ISG could be eluted by raising the NaCl concentrationto1.35M.Afterextensivedialysis, the eluatewasconcentrated by lyophilization. The concentrated material was applied to a 6%

SDS-polyacrylamide gel. ISG was eluted from the gel by diffusion.

Typically, the yield was200-300Agofhomogenous ISG.

Proteolyticdigestion andseparation ofpeptides

180AgISG were dissolved in 400A150mMTris-HCI(pH8.0)anddigested with 3Agoftrypsinat37°C. After 3 h,anadditional 3Agoftrypsinwas added anddigestionwascontinued for 6 h.Peptideswerefractionatedby reversedphase HPLCon aLichrosorb RP 18, 10Amcolumn(E.Merck, Darmstadt, FRG)asdescribedby Ertl etal.(1989). Deglycosylationof ISG andsubsequentproteolyticdigestionwereperformedasdescribed for the glycoprotein SSG 185by Ertletal. (1989).

Generation ofacDNA probebyPCR

RNA and poly(A)+ RNA were extracted from Volvox spheroids as describedby Kirk and Kirk(1985). Reversetranscriptionwasperformed inafinal volume of20 11containing 50mMTris-HCI(pH8.3),40mM KCI, 6mM MgCl2, 1 mMdithiothreitol,20 URNAguard(Pharmacia), 150pmol antisenseoligonucleotide (GGG/T AGGTTNCCGAA G/ATT), 1mMeach dNTP, 250 ngpoly(A)+RNAand 200 U of MoMuLVreverse transcriptase.Incubationwas at42°Cfor45 min.After addition of 80Asl

of1xPCR buffercontaining 500pmolofsenseoligonucleotide (GTNTA T/CAAT/CAAT/C ATT/C/AGC)and2.5 U ofTaq polymerase,thermal cycling(Perkin-Elmer cycler480)wasinitiated(30 cycles:denaturation at94°Cfor 45 s, annealingat50°C for30sand extensionat72°C for 45 s).The resulting360bpDNAfragmentwasligatedinto theSnaIsite ofpUC18andsequenced.

Cloningof theISGgene

The V.carterigenomic libraryinXEMBL3(Frischaufetal., 1983)described byErtIetal.(1989)wasusedtoclone the ISG gene. Thescreening procedure followed standard techniques (Sambrooketal., 1989). DNAsequencing wasperformed by the chain termination method(Sangeretal., 1977)using T7 DNApolymerase (Pharmacia). Synthetic oligonucleotideswere used to sequencethe ISG gene inboth directions.

PCRwith RNA from asingle embryo

Synchronously growingVolvoxspheroidscontainingpreinversion embryos were disrupted byforcing them througha 0.5 mm hypodermic needle.

Somatic cell sheetswereremovedbyfiltration(100 ,smmeshnylon cloth).

Asingle embryowasthen identified under thestereomicroscope,picked up withadrawnplasticpipetteandtransferred into 10Alsterilelysisbuffer (50mM Tris-HCI, pH8, 300mM NaCI,5 mM EGTA and2% SDS) containing 1

1tg

Ecoli SS RNAascarrier. After 10 minat28°C,RNA wasprecipitated with 30A1ethanol. The washedprecipitate(70%ethanol) wasdissolvedin 10Alreversetranscriptionbuffer and furtherprocessed asdescribed above. The senseandantisense oligonucleotidesusedwere

5'-GATGGGTTCGCGTAGGCTCG (exon1)and5'-GGTCAGGCCTT- CCGTAATCCAGTA(exon2),respectively.35cyclesof PCRamplification

were performed (94°C,45 s; 60°C, 30 s; 72°C, 45 s).

Electronmicroscopy

ISG andsubfragmentswererotaryshadowed withplatinumand visualized asdescribed byMorgelin etal. (1988).

Preparationof recombinant ISG polypeptide

AcDNAfragmentencodingaminoacid positions5-172 wasgenerated by PCR and subsequently cloned into pETl1 and expressed inE.coli accordingtothe method of Studieretal. (1990).The recombinantpeptide wasoverexpressed and located ininclusion bodies. The isolated inclusion bodieswererepeatedlywashed with4 M ureaandsolubiizedinSDSsample buffer.Onlyasingle major bandwasdetectedon anSDS-polyacrylamide gel(12%); this materialwaseluted,concentrated inamicroconcentrator (Centricon 10, Amicon)and usedtoraisepolyclonalantibodiesinrabbit.

technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43).

References

Adair,W.S. andAppel,H. (1989)Planta, 179, 381-386.

Adair,W.S., Steinmetz,S.A., Mattson,D.M., Goodenough,U.W. and Heuser,J.E. (1987)J. CellBiol., 105, 2373-2382.

Cassab,G.I. and Varner,J.E. (1988) Annu. Rev. Plant Physiol., 39, 321 -353.

Catt,J.W., Hills,G.J. andRoberts,K. (1978) Planta, 138,91-98.

Dubois,M., Gilles,K.A., Hamilton,J.K. and Rebers,P.A. (1956)Anal.

Chem.,28, 350-356.

Ertl,H., Mengele,R.,Wenzl,S., Engel,J.andSumper,M. (1989)J. Cell Biol., 109, 3493-3501.

Frischauf,A.-M.,Lehrach,H., Poustka,A.andMurray,N.(1983)J. Mol.

Biol., 170, 827-842.

Goodenough,U.W. andHeuser,J.E. (1988)J. CellSci., 90,717-733.

Green,K.J., Viamontes,G.I. and Kirk,D.L. (1981)J. Cell Biol., 91, 756-769.

Holtfreter,J. (1943)J. Exp. Zool., 94, 261-318.

Holtfreter,J. (1944)J. Exp. Zool., 95, 171-212.

Kelland,J.L. (1977)J. Phycol., 13,373-378.

Kirk,M.M.andKirk,D.L. (1985)Cell, 41, 419-428.

Kirk,D.L., Birchem,R.andKing,N. (1986)J. CellSci., 80, 207-231.

Morgelin,M.M., Paulsson,M., Hardingham,T.E., Heinegard,D. and Engel,J. (1988)Biochem. J.,253, 175-185.

Mort,A.J. andLamport,D.T.A. (1977)Anal. Biochem.,82,289-309.

Pickett-Heaps,J.D. (1970)Planta,90, 174-190.

Provasoli,L.andPintner,I. J.(1959)InTyron,C.A.andHartnan,R.T.(eds), TheEcology ofAlgae. SpecialPublicationno.2, Pymatuning Laboratory of FieldBiology, UniversityofPittsburgh, Pittsburgh, pp. 84-96.

Roberts,K. (1974)Phil. Trans. R. Soc. Lond. B, 268, 129-146.

Roberts,K.,Grief,C. andShaw,P.J.(1985)J. CellSci.,Suppl. 2, 105- 127.

Sambrook,J., Fritsch,E.F.andManiatis,T. (1989)MolecularCloning:A LaboratoryManual,2nd edition. ColdSpringHarborLaboratory Press, Cold Spring Harbor, NY.

Sanger,F., Nicklen,S. andCoulson,A. R. (1977)Proc. Natl. Acad. Sci.

USA, 74, 5463-5467.

Schlipfenbacher,R.,Wenzl,S., Lottspeich,F.andSumper,M.(1986)FEBS Lett.,209, 57-62.

Showalter,A.M., Bell,J.N., Cramer,C.L., Bailey,J.A., Vamer,J.E. and Lamb,C.J. (1985) Proc. Natl. Acad. Sci. USA, 82,6551-6555.

Starr,R.C. (1969)Arch. Protistenk., 111,204-222.

Starr,R.C. (1970) Dev. Biol., Suppl. 4, 59-100.

Starr,R.C. and Jaenicke,L. (1974) Proc. Natl. Acad. Sci. USA, 71, 1050-1054.

Studier,F.W., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990) Methods Enzymol., 185, 60-89.

Tschochner,H., Lottspeich,F. and Sumper,M. (1987) EMBO J., 6, 2203-2207.

Viamontes,G.I. andKirk,D.L. (1977)J. CellBiol, 75, 719-730.

Viamontes,G.I., Fochtnann,L.I.andKirk,D.L. (1979)Cell, 17,537-550.

Wenzl,S. andSumper,M. (1982)FEBSLett., 143, 311-315.

Wenzl,S. andSumper,M. (1986a) Cell,46, 633-639.

Wenzl,S. andSumper,M. (1986b)Dev. Biol., 115, 119-128.

Woessner,J.F. andGoodenough,U. (1989) PlantCell, 1,901 -911.

ReceivedonJanuary 30, 1992; revisedonFebruary 27, 1992

Fluorescencemicroscopy

V.carteri spheroids were disrupted by forcing them througha0.4 mm

hypodernicneedle. Thesubsequent procedurewas asdescribedpreviously (Wenzl and Sumper, 1986a).

Acknowledgements

We wish to thank Dr J.Engel for taking the electron micrographs, Dr R.Deutzmann for sequencing peptides and Elke Berg for excellent