adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

Probing

an

adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

A.Noegel, C.Harloff, P.Hirth, R.Merkl, M.Modersitzki, J.Stadler, U.Weinhart, M.Westphaland G.Gerisch

Max-Plank-Institut fuirBiochemie, D-8033 Martinsried beiMunchen, FRG Communicated by G.Gerisch

Expressionof developmentallyregulated membraneproteins ofaggregating cells ofDictyosteliumdiscoideum is subject to several control mechanisms. One ofthem involves periodic cyclic-AMP pulsesassignals forgeneexpression. Toincrease the probabilityofselectingmutantsspecifically defective in the contactsiteA(csA)glycoprotein, oneof the characteristic proteins of aggregatingcells, we havebypassedtherequire- mentfor bothcyclic-AMP pulsesand another control element by two runs of mutagenesis. A 'double bypass' mutant, HG592,wasobtained whichaggregated in nutrient medium where wild-type did not develop. Mutants defective in ex- pressionofthecsA-glycoproteinwereselected from HG592 by fluorescence-activated cell sorting and colony immuno- blottingusingamonoclonalantibodyspecific for that protein.

One among 51

csA-negative

mutants,

HG693, specifically

lacked thecapability of forming EDTA-stable intercellular contacts. Itacquired chemotacticresponsiveness and devel- opedinto fruiting bodies. Expression of the transcripts for eight developmentallyregulated proteinswasdeterminedin HG693. Seven oftheRNAspecieswerenormally expressed;

theywererecognized by cDNA cloneswhich had beenpro- duced from poly(A)+ RNAisolatedfrom membrane-bound polysomes. The singleRNA species whichwasnot substan- tially expressed inHG693 wasrecognized bya cDNA clone thatwasobtainedbyscreeninga

Xgtll

library withananti- body

specific

forthecsA-glycoprotein. When probingRNA from wild-type cells, this clone hybridized with a single developmentally regulatedRNAspecies of 1.9 kbwhoseex- pression wasstrongly enhanced by

cyclic-AMP

pulses.

Ap-

pearance ofthis RNA coincidedwith the expression ofthe csA-glycoprotein.

Keywords:cell adhesion/contact sitesA/bypassmutants/cyclic- AMP signalsIDictyostelium discoideum

Introduction

During early development of Dictyostelium discoideum, cells become capable of producing cyclic-AMP signals and of re- sponding to them by chemotactic orientation (Bonner et al., 1969). In addition, aggregating cells form EDTA-stable inter- cellularcontactsandassemble intostreamsofelongatedcells dur- ingtheirmovementtoaggregationcenters (Beugetal., 1973).

The onset of chemotaxis and the acquisition of EDTA-stable adhesivenessareaccompanied by the increasedexpression ofa number of membrane proteinssuchasadenylatecyclase, cyclic- AMPphosphodiesterase, cyclic-AMPreceptors and thecontact site A (csA) glycoprotein. With the

exception

of

adenylate

cy- clase, theseareplasma membrane

proteins exposed

ontheouter

surface. The

csA-glycoprotein

is an

integral

membrane

protein

with an apparent mol. wt. of 80 kd which is expressed at the sametimeasthe cellsacquirethecapabilityofformingEDTA- stable contacts(MullerandGerisch, 1978).Cyclic-AMPpulses stimulate the expression of the above-mentionedproteins.Cells ofthe AX2strain of D. discoideum produce pulses ofcyclic AMP autonomously by periodic activation of adenylate cyclase (Roos etal., 1977) and respond tocyclic AMPviacellsurfacerecep- tors. Inanotherstrain,AX3,theproductionofcyclic-AMP sig- nals is insufficient to carry on normaldevelopmentinsuspension cultures. Insuch cultures AX3 cells become fully aggregation competentonly if they are stimulated by applied pulses of cyclic AMP (Chisholm et al., 1984; Gerisch etal., 1984). Pulsatile stimuli are required for enhancement of development because the response system adapts to steady concentrations of cyclic AMP, a process which is accompanied by phosphorylation of thecyclic-AMP receptors (C.Kleinetal., 1985; P.Klein etal., 1985; Devreotes andSherring, 1985). Phosphodiesterase is an essential component of thesignal system since it degrades cyclic- AMPbetween thepulses and thus allows the response system tode-adapt. Early development is inhibited in a phosphodiester- ase-negativemutant(Brachetetal., 1979) and in wild-typecells which are kept in theadapted state by exposure to a low steady- stateconcentration ofcyclic AMP (Gerisch et al., 1984).

Toanalyse the system that controls earlydevelopmentup to theaggregationstage,and toinvestigate thefunctionof proteins whoseexpression is coupled to that system, we have isolated mutants in which some of the controls are bypassed. Mutants which do not require pulsatile cyclic-AMP signals for their developmentwereisolated by screening mutagenized cells of the AX2strain on agar containing3',5'-cyclic adenosine phosphoro- thioate (cAMPS) (Rossier etal., 1980). Because cAMPS is a phosphodiesterase-resistantagonist of the cyclic-AMP receptors it inhibitswild-typedevelopment, asdoes a low steady-state con- centration ofcyclicAMP(Rossieretal., 1978). Mutantswhich aggregate inthe presence of cAMPSwerecollected and one of them, HG302, waschosenfor furtheranalysis (Wallraffetal., 1984). Inthat mutant thecsA-glycoproteinis still under stringent developmentalcontrol. Thisfindingsuggeststhat a second control system exists which suppresses the csA-glycoprotein during growth and enables its expression in starved cells. HG302, in

turn,

wasmutagenized anda 'double-bypass' mutant, HG592, was isolated which aggregated in nutrient medium (Gerischet al., 1985b). Wild-type

cells

need to betransferred to non-nutrient bufferinorderto develop. They do not expressthecsA-glyco- protein as long as they remain in nutrient medium, even after entering thestationary phase. HG592 expresses thecsA-glyco- protein in nutrient medium but notbefore the end ofgrowth, indicatingthat there is at leastonecontrol stepnotbypassed in this mutant, and that this step prevents the

csA-glycoprotein

from beingconstitutively expressed.WemutagenizedHG592 cells for the selection of mutants defective in expression of the csA-glycoprotein by cell-surface labeling with a csA-specific monoclonal antibody and fluorescence-activated cell sorting.

UseofmutagenizedHG592cellsfortheselection ofmutants

© IRLPressLimited, Oxford, England 3805

defectiveincertain

developmentally regulated proteins

prevents partofthe

pleiotropic

mutants frombeing selectedwhich donot developbecause of adefectin someof the

regulatory

genes. Thus the

probability

is increased of selecting a mutant which is

specifically

defective inthe

csA-glycoprotein.

Inthispaperwe describe a mutant obtained from HG592,

HG693,

in which a defectin

EDTA-stable

celladhesionisassociatedwithadefect in csA expression. We have

probed

the

transcripts

of eight

developmentally

regulated genes using cDNAclonesand show that HG693 is notgenerally blocked in development.

Results

Selection ofmutant HG693 defective in the

csA-glycoprotein

Mutagenized cells of the double bypass mutant HG592 were grownfor aboutfour generations innutrient mediumand kept inthatmedium until

non-mutated

cellshad

expressed

the csA-

glycoprotein

ontheirsurfaces.Thecells were thenincubatedwith mAb71, anantibody

recognizing

anexternal

portion

of thecsA-

glycoprotein,

and

subsequently

labeled with

FITC-conjugated

anti-mouse IgG. Cells showing weakor no fluorescence were selectedbyacellsorterandgrownfor ^- 10

generations.

After developmentto aggregation competencethe cells were labeled againwithantibody and sorted, and wereclonedintoalawnof bacteriaonnutrientagar plates.Colonieswereblottedontonitro- cellulose filters and labeled with

[125I]mAb

71. Out of 803 selected clones, 51 colonies, i.e., 6.3%, showed ^nodetectable labeling with theantibody. Except foroneclone, these mutant clonesshowednoaggregation oronlylooseassembliesofcells which didnotdevelopfurther.Becausedevelopmentwasgeneral- lyblockedinthese mutants weassumethattheyweredefective in regulatory genes whose activities were still essential for development of theprogenitor strain, HG592. The

only

csA- defective mutant selected which showed

aggregation

and pro- ceeded with development into fruiting bodies was HG693 (Figure 1).

7hedefectin

csA-glycoprotein

expressionis

paralleled

byadefect in EDTA-stable cell adhesion

Toprovethat theentire

csA-glycoprotein

and notonlyanepitope on it is lacking in mutant HG693, twomonoclonal antibodies

produced

by

independently

isolated

hybridomas

were

applied

in additiontomAb71.Evidencehasbeen

provided previously

that all three antibodies aredirected againstthe

polypeptide

moiety of the

csA-glycoprotein

(Bertholdtetal., 1985). Resultsobtain- ed with mAb 294 are shown in Figure 2. Neither in starved

unstimulated

cells nor after stimulationby

cyclic-AMP

pulses for 23 hwasthe

glycoprotein

detectablewith this

antibody,

and alsowith mAb448no

csA-glycoprotein

wasdetected.InHG592, theparentstrain of HG693,the

csA-glycoprotein

was

recognized

bybothantibodies, asitwaswithmAb 71. The

csA-glycoprotein

wasalreadyweaklyexpressedinHG592cells at the time oftheir harvest fromnutrient medium, inaccord with

previous

results

(Gerisch

etal., 1985b),and theexpressionwasstronglyincreased during development (Figure2). Toexcludethe

possibility

that the

glycoprotein

was degraded during sample

preparation

by

overproduction

of a protease in HG693, and also to exclude thepresenceof any inhibitor ofantibody binding in thisstrain, equalamounts ofHG592andHG693weremixedand

subjected

togetherto

SDS-polyacrylamide

gel

electrophoresis

ofthepro- teins. As shown inthe last lane ofFigure 2no

degradation

of the

glycoprotein

from HG592 was observed in the mixture.

ThefindingthatHG693was notgenerally blockedindevelop- ment

prompted

ustodetermine cell functions affectedincoinci-

3806

2cm

id'.. ;t *

AI1 w

.0,

r^{f~ A}

_* *\ *U

.._I i

p.

1...

A

* 4

I I

'"'tlI '. ;

Fig. 1. Colonyblots andaggregatesonagarplates ofthe double bypass mutantHG592 (left panel)and thecsA-defective mutantHG693 (right panel). Top: autoradiogramofacolonyimmunoblot labeled with

(1251]mAb

71. OnlytheaggregatesofHG592 arestrongly labeled. Middle: proteinsof thesameblotstainedwith Ponceau S. Bottom:aggregatesandfruiting bodies.

dencewith thedefectin

csA-glycoprotein expression. Figure

3 showsthat

aggregation-competent cells

ofHG592 formed EDTA- stablecontacts, whereasthecell-to-cell adhesionofHG693was

strongly sensitiveto10 mM EDTA.

Figure

3furthershows that HG693 cells

acquired

the

elongated

shape

typical

oftheaggre- gation stage. Another

developmentally regulated

cell function involvedincell

aggregation

is the

chemotactic

reponseto

cyclic

AMP(Bonneretal., 1969). Figure4shows that cellsofHG693 developed

chemotactic responsiveness

to this attractant.

Normal expressioninHG693ofseven outof

eight

development- ally

regulated

RNA species tested

Developmentally

regulated membrane proteins playkey roles in the

cyclic-AMP

signal systemand incelladhesionof

aggregating

cells.We havetherefore isolated

cDNA-probes

for

appropriate

genesandtheirtranscripts.^Onestrategyusedincludedthecloning

1.

Dictyostelium

-HG592--1--- HG693--

HG592

+

HG693

0.6 E , 0 4

X3O ~r~O..&o-OO{C\ O 02

-6A-2 A X &.- -A-A 0.

C t0 20 30 40 50 min

,-

C C 6P C

0 6 0 6 6i 6 _

Fig. 2. Autoradiogram ofimmunoblotted proteins separated by SDS- polyacrylamide gel electrophoresis. Cells ofmutants HG592orHG693 ^were harvestedeither from nutrient medium (0)orafter 6 h of starvation (6) with (P) orwithout (C) stimulation by pulses of cyclicAMP. Total cellular protein equivalentto 1 x 106 cellswasapplied perlane; in the last lanethe

equivalent of 5 x 105cells of each mutantwas applied.

II

ofpoly(A)+ RNA from membrane-bound polysomes of cells harvestedatthe6-hstageofdevelopment, the beginning ofag-

gregation competence. The cDNA library was differentially screened with DNA complementary to poly(A)+ RNA from membrane-bound polysomes of the 6-hstageofdevelopment and with DNAcomplementary tototal cytoplasmic poly(A)+ RNA from growth phase cells. Seven different cDNA clones which hybridized preferentiallyorexclusively with the probe from the 6-hstagewereused in thestudy presented here. A secondstrategy

was to screen acDNA library in the Xgtl expression vector withantibodies specific for the polypeptide backbone of the csA- glycoprotein. Herewehave used clone Xc523 coding forapoly- peptide recognized by mAb 294. Northern blots of minigels in- dicate that thestringency of regulation during early development ofthe AX2 strain varied between the RNAspecies (Figure 5).

In the bypassmutant HG592 all RNA species were expressed atthe 5-h stage, asthey werein the AX2 wild-type strain, and five ofthemwerealready strongly expressed when thecells were

harvested from nutrient medium. This observation reflects the bypassing ofdevelopmental controls in HG592.

In mutant HG693 seven of the RNAspecies accumulatedto similar levels ^asinthe AX2 and HG592 strains. Theonly RNA species that behaveddifferently wasthat recognized by ,uc523.

Onlyinsignificant labelingwasobtained withRNAof themutant under conditions whichgaveastrongsignalwith RNAfromag-

gregation-competent cells ofthe AX2 and HG592 strains.

Therecognition by mAb 294of Xgtl1-hybrid phagescarrying c523 DNAsuggested thatthisinsert contained part of thecoding

sequences forthe csA-protein. This assumptionwas supported by the stringentdevelopmental regulationofthetranscripts. Since thecsA-glycoprotein isknowntobeweakly expressedinsuspen- sioncultures of starved AX3 cellsandtobedrastically induced

100

Jrm

HG592 HG693

Fig. 3. Cell agglutination inmutants HG592and HG693. Top: recoid of light scattering monitored inanagglutinometer. Ordinate: light scattering values Earearbitraryunits; low values indicate that cells have formed agglutinates. Abscissa: time in minafter transfer of the cells tothe agglutinometer. Cells ofmutantHG592 (A,A)and HG693 (0,0) were

harvestedat6hof starvation in 17 mMphosphate buffer, pH6.0. During starvation the cells had been stimulatedby pulsesofcyclicAMP.

Subsequentlythe cells werewashed andsubjectedtotheagglutinometer either with 10mMEDTA (A, 0)orwithout EDTA (A,0)addedtothe phosphate buffer. Middle: photographs of cellsandagglutinates ofHG592 (left) and HG693 (right) after ¹ hwithout EDTAintheagglutinometer.

Bottom: thesamewith 10mM EDTA.

116- 93^- 64-

45 -

kd

-0 4W-&-Al.'*- -0 -4& 4 0-4 40

'.. I -1

I.' 1.t

.4 1. %

C.. I

.!, le.l

1 s

V .

I

.i _5 -- 'V) .- 41i

.. 9

Fig.4. Chemotactic response of HG693 cells to cyclic AMP. The cells werestimulated by a micropipette filled with 1O-3 M cyclic AMP, and photographs were taken at the times indicated after positioning of the micropipette.

9' 9,

co ',tS;co ⁽⁰

Q.

44 ^_!

Zm '-

; v_..

44;:

p

*.4

a *rX 2

L.

p

Fig. 6. Northernblots of totalcytoplasmicRNA of D. discoideum strain AX3. TheRNA washybridized with the same cDNA probes as in Figure 5. The cells were harvested from nutrient medium(0) or after starvation for 4 h(4)or6 h(6) with (P) or without (C) stimulation by pulses of cyclic AMP.

in these cells by pulses of cyclic AMP (Gerisch et al., 1984, 1985b), wehaveexaminedregulation of the RNA species, includ- ing the onehybridizing with

p523,

in stimulated andunstimu- latedAX3 cells (Figure 6). As in the AX2 strain, in the AX3 straindifferences wereobserved among the RNA species in the stringency of theirdevelopmentalregulation (Figure 6). For ex- ample, RNA recognizedby cloneAl1B6 was already substan- tiallyexpressedin growthphase cells andsteadily accumulated during development, whileRNAspecies hybridizingtoP29F8, M7E5 or

p523

werenotdetectablyexpressed during growth and accumulated only slightly during the developmentofunstimu- lated cells. Thestimulationofexpressionof thesee RNA species bycyclic-AMP pulseswascorrelated with thestringencyof their regulation. Expression of the RNAhybridizing to clone

Al

1B6 wasonly slightly enhanced by the cyclic-AMP pulses, whereas the RNAhybridizing toclone

p523

was drastically induced by the stimulation.

-i, 'Vr2'1-*1!>-',

Properties of

z cDNAclone Xc523 isolated

by labeling plaques

with

H G5 9 2

the csA-specific

mAb

294

Phage Xc523 carried a 1.3-kb insert, c523, that recognized in Southernblots of DNA from the AX2 strain one EcoRI frag- ment of 6.8 kb and two

HindIII

fragments of -4.9 kb and

>10kb,respectively.Northern blots showed that thec523 DNA

hybridized

with a

single

RNAof 1.9 kb.

Figure

⁷ showsmore 9 * , precisely* z z

z

thanFigure5 that theRNArecognized byc523^was

I,

^wnotdetected

^{during growth}

^andwas

strongly expressed

inAX2

cellsat5 h ofstarvation

(Figure 7). Only

a

faintly

labeled band was seenwith RNA ofHG693 cells that were starved for 5 h with or without stimulation by cyclic-AMP pulses. The RNA ,

5-,

',''0-:)5

-9

-_ ';t , recognized by

c523

might be slightly larger in HG693 than in

H

G 6 9

.>: AX2.

Fig. 5. Northernblots of totalcytoplasmicRNA of D. discoideum strain AX2(top), HG592(middle)and HG693 (bottom). The cellswereharvested fromnutrient medium(0)orafter 5 h of starvation (5), and theRNAwas hybridizedwitheight different cDNA probesasindicated.

3808

Discussion

The goal of the workpresented here was to relate a defect in the expression of thecsA-glycoproteinto afailure of a specific

0 mrn b mlr" 30*.mS

5,--1_

*: ...

I

.1

v

50 rri

*.~ ~ ~ ~ " Js!4 6, r-

ci,. F.. p)'- 3

ov

f

Contact site AmutantinDictyostelium

Start I AX2-,r--

FG693i-i

4.1 ^-

1.9

^-

kb

c

0 5

C

p

0 5 5

Fig. 7. Northern blot of total cytoplasmicRNA from AX2 and HG693 cells harvested either fromnutrient medium (0) orafter⁵hof starvation (5) with (P) orwithout (C) stimulationby pulses of cyclic AMP. The location of rRNAs (4.1 kb and 1.9 kb) is indicated.

cell function. The large number of regulatorygenesinvolvedin D. discoideum developmentmakesitatedious tasktoselectstruc-

tural genemutants forproteins which are, like the csA-glyco- protein, under the control ofthese genes. Previous attempts to select csA-defective mutants from mutagenized wild-type cells yielded onlypleiotropicmutantswhosedevelopmentwasblocked prior^tothe expression of thecsA-glycoprotein. Mutantsselected byuseofacarbohydrate-specific antibody (Murrayetal., 1984),

werepartiallydefective inglycosylationbut expressed the csA- protein (Gerisch et al., 1985a).

Inthepresentstudyadoublebypassmutantwasusedas apro-

genitor, and one mutant was obtained which appearedto lack specifically the csA-glycoprotein. Since this mutant, HG693, showedchemotaxistocyclicAMP andunderwentdevelopment beyond the aggregation stage it didnot suffer froma defect in the overall controlofdevelopment. Theinabilityofaggregating cells ofHG693 toformEDTA-stable cell contacts is in accord withpreviousfindingsindicatingthatEDTA-stablecell adhesion isblocked by Fabfragmentsfromantibodies directedagainstthe csA-glycoprotein (MullerandGerisch, 1978).Theabilityofthe

mutant toform

fruiting

bodies agrees with other

findings

accord-

ing

towhich the

csA-glycoprotein

is

degraded during

the multi- cellularstage

following aggregation, suggesting

that thefunction of this

glycoprotein

islimitedtothe

aggregation

stage. A ques- tion that remainsto be answered is the

relationship

ofEDTA- stable adhesiveness to the formation of streams

by

cells ag-

gregating

on asubstratum. Previous studieshave shown that the end-to-end adhesion of

streaming

cells isEDTA stable

(Beug

et

al., 1973).

On theother

hand, long

streams areformed

by

amu- tant

exhibiting

a defect in

carbohydrate synthesis.

Thismutant

produces

greatlyreducedamountsofa

partially

glycosylatedcsA-

protein

and

accordingly

exhibits weaker EDTA-stable adhesiveness

(Murray

et

al., 1984;

Gerischet

al., 1985b).

HG693 also

forms

streams. Furtherworkmust

clarify

whether thisstream formation is due to the EDTA-sensitive type of cell adhesion which is

independent

ofthe

csA-glycoprotein,

whether small amountsof the

csA-glycoprotein

notdetected

by

theimmunoblot-

ting procedure

are

synthesized

inthe mutant, orwhether weak EDTA-stablecelladhesion

barely

detectedbythe

agglutinometer

assay used can be mediated

by

cell surface components other than the

csA-glycoprotein.

Examining

the

expression

of

eight

different

developmentally regulated poly(A)

+ RNA

species

showed thatonlyoneRNAwas

strongly

suppressedinmutantHG693. The cDNA cloneXc523 which

recognized

the

suppressed

RNAwasselectedfromaXgtl1

library by labeling plaques

with mAb

294,

an antibody

highly specific

for the

polypeptide moiety

of the

csA-glycoprotein (Figure 2). Compared

with the RNA

species

recognizedby the othersevencDNA

probes,

which varied in thestringencyof their

developmental regulation,

the RNA

hybridizing

tocloneXc523

belonged

to the most

stringently

regulated ones. Both this

transcript

and the

csA-glycoprotein

wereundetectable in

growth phase

cells and were expressed in AX2 cells at 5 h ofstarva- tion. TheRNA remained almostunexpressedin starvedcells of strainAX3cultivated in

suspension

andbecamestronglyexpress- ed inthese cells after stimulation

by pulses

of

cyclic AMP,

in coincidencewith

expression

ofthecsA-glycoprotein (Gerischet

al., 1984,

1985b). Together, these results suggest that clone Xc523containsaninsert

homologous

tothe

coding region

ofthe

csA-protein

gene. The size of the RNA of 1.9 kbrecognized

by

thisinsert is sufficienttocode foraproteinofmol. wt. 53

kd,

the presumed size of the polypeptide moiety of the csA- glycoprotein (Hohmann et al.,

1985).

The selective andsubstantial suppression of RNA

hybridizing

with cloneXc523 and the apparent presence of minuteamounts ofthis RNA suggest thataregulatory regionofthe

csA-protein

gene is

changed

inHG693. Final proofinfavour ofor

against

thisconjecturewill beprovided by sequencing the

csA-protein

genes from the wild-typeand mutant.

Materialsand methods

CultureofD. discoideum strains

Cellswerecultivatedat23 °C in nutrientmediumwith 1.8%maltose asdescribed byWattsandAshworth(1970).Cells of strains AX2 (clone214)andAX3were harvestedatdensitiesofnot morethan5 x 106cells/ml. Cellsof mutantsHG592 andHG693,which grew in the medium to maximal densities of 3-5 x 106/ml, wereharvestedatdensitiesof not morethan 2.2 x 106/ml. Developmentwas initiatedby washingcells in 17 mMSoerensenphosphatebuffer,pH6.0('non- nutrientbuffer').The cellswereresuspended in the same buffer at a density of 1 x 107/ml,andagitatedonarotaryshaker at 150 r.p.m. for AX2 and AX3 andat220 r.p.m. forHG592andHG693. Cells were allowed todevelopeither withoutstimulation,orwithstimulation bycyclic-AMP pulses of 20 nMampli- tudeappliedevery 6 min.

Foraggregation on agar and forcolony blotting cells were cultivated with 3809

Escherichia coli B/2 on nutrient agar containing 0.1% bacteriological peptone (Oxoid), 0.1% glucose and 2% Bacto-Agar (Difco) in non-nutrient buffer.

Preparation of antibodies

Monoclonalantibodies 33-294-17, 41-71-21 and 41-448-9 (Bertholdtetal., 1985) arereferredto asmAb294, mAb71 andmAb448, respectively. Antibody IgG was purified from hybridoma culturesupernatants on protein A-Sepharose columns.

For iodination, 70-100tigIgG were labeled in a total volume of200

/d

con- taining phosphate-buffered saline, pH 7.2,0.5mCi of[125I]iodide(IMS30,Amer- sham), and 15 ,ug chloramineT. After 45s at room temperature, 100,1Iof a saturatedtyrosinesolution was added andthe[1251]IgG separatedwith Dextran Blue and Phenolredon a10mlcolumn ofSephadex G50 medium.Forlabeling of blotstheIgG wasdiluted to 105-106c.p.m./ml.

Mutant selection andimmunoblotting

Growthphase cells of HG592 were mutagenized by incubating 1 x 108cells in5ml of17 mMphosphatebuffer, pH 7.0, with 8 mg of1-methyl-3-nitro-l- nitrosoguanidine for 20min at roomtemperaturein the darkundergentleagi- tation. Thesurvivalrate was8%.The cells werewashed, dispensedinto 10flasks with 30mlofnutrient medium, and shakenat23'C for 5days. At thattime HG592 hadexpressed thecsA-glycoproteinonthecell surfaces. 2 x 106 washed cellswereincubated for 15 minat -4°Cunderheavy shakingin 150 1Inon- nutrient buffercontaining 15 jig of mAb 71. The cellswerewashed and labeled underthesameconditions with 50-fold dilutedFITC-conjugated sheepanti-mouse IgG(InstitutePasteurProduction).The labeledcellswerewashed innon-nutrient buffer, resuspended in phosphate-buffered NaCl (150 mM), pH 7.2, filtered through fine nylon gauze, and sorted in the coldusinga FACSIVcellsorter.

The 2% fraction of the cell population with the lowest fluorescenceintensitywas selected.Perflask ofmutagenized cells3 x 106 cellsweresorted and the selected cellsgrown inasuspensionof5mlof1 x 1010bacteria perml ofSalmonella minnesotaR595(Gerischetal., 1985a). After2days,25 ml more of the bacteria suspensionwereadded.After 5daysthe cellswerewashed in non-nutrient buf- fer and starved for 6h with stimulationby pulsesofcyclicAMP. The cellswere again labeled with mAb 71 andfluorescentantibody for sortingasdescribed, andweredirectly clonedusingasingle-cell depositionsystemontobacteria-coated nutrientagarplates(Francisetal., 1985). Colonieswereblotted ontoBA 85 nitrocellulosefilters(SchleicherandSchull,3354Dassel, FRG),frozenonametal plate cooledby dry ice, thawed and washedasdescribedbyStadleretal.(1984) forimmunoblotting.Afterlabelingwith[125I]mAb71 andautoradiography,blots werestained with0.2%PonceauS(Cat.No.33429, Serva, Heidelberg)in3%

trichloroacetic acid(TCA)andwashed in the TCA solution. Theoriginalplates

werekept in thecold;enoughcells remainedontheagarsurface afterblotting to startcultures frommutantsidentifiedbyimmunoblottingascsA-defective. Cells of these mutants were recloned beforeuse.

SDS-polyacrylamidegelelectrophoresiswasperformedin10%gelsaccording toLaemmli(1970). ImmunoblotswereobtainedaccordingtoTowbinetal.(1979) asdescribed byBertholdtetal. (1985).

Assaysforcell adhesion and chemotaxis

Cell adhesionwasquantitated byuseofamicroprocessor controlled version of theagglutinometer described by Beug and Gerisch(1972)inwhichlightscatter- ing could be recorded continuously. Cuvettes containing1 x 107 cells/mlwere rotatedat40 r.p.m. toexpose the cellsto constantshearforces. The decrease inlight scattering duetocell aggregateformationunderthese conditionswasus- edas a measure of cell-to-cell adhesion.

Forthe assay ofchemotaxis, HG693 cellswerestarved for 5h, washed with non-nutrientbuffer and transferredtothe Teflonsurface ofaPetriperrndish(Her- aeus,6450Hanau, FRG). Micropipettesfilled withcyclicAMPsolutionwere used as describedby Gerischand Keller(1981).

cDNAcloning

Seven cDNAcloneswereobtainedasdescribedpreviously usingpoly(A)+RNA frompolysomesbound toendoplasmic reticulum membranesofcells harvested at6 h ofstarvation(Gerischetal., 1985b). The plasmidused wasp2732Bcon- structedby J.D.Monahan, Roche Institute, Nutley, NJ, and the E. coli host strain wasBJ5183(recBC-sbcB-) (Hanahan, 1983). PlasmidDNA wasisolated ac- cordingtoBimboimandDoly(1979) and nick-translated inserts were used as probes inNorthern blots. CloneXc523wasobtainedby screening with mAb 294 aXgtl1library providedto usbyDrRichardKessin, Columbia University,NY.

Thelibrary made by Dr M.-L.Lacombe was afulllength cDNA library from RNA that hadbeen inducedby cyclicAMP. Forscreening104 phages per 12 x 12 cmplatesweregrownon E. coliRY1090 (Young and Davis, 1983). After 3 hat43°Cnitrocellulose filterspreviouslysoaked in 10mMIPTGwerelaid ontotheplatesand incubationwascontinuedat37°Cfor 14-16 h. The filters wereextensively washed with buffer containing 10mMTris, pH 8.0, 150 mM NaCl, 0.05 % Tween20and0.02%azide. Theantibodywasiodinatedasdescribed and thefilterswereincubatedfor 2 hwith [1251]mAb294, 105-106c.p.m./ml, in theTris-NaCI-Tweenbuffer. Afterwashingseveral times with thebuffer,the filterswereexposedtoKodak X-OmatARfilm.PhageDNAwasisolated accord-

3810

ingtoManiatisetal. (1982) usingRY1088ashoststrain,andthe insertDNA c523 ofphageXc523wasrecloned intotheEcoRI site of theplasmidvectorgemini 2using DHIashost strain (Hanahan, 1983); theplasmidobtainedwasp523.

DNAof theplasmid wasisolatedaccordingtoHolmes andQuigley (1981).

Isolation andhybridization ofRNA andDNAfromD. discoideumstrains RNAwasextracted from cellsattheindicatedstageswithphenol-chloroform.

Northern blotswereobtained either fromminigelsof6cmlengthor,foradetailed analysis,fromgelsof 20cmlengthcontaining1.2%agaroseand 6%formaldehyde according^toManiatisetal.(1982).Ifnotindicatedotherwise,inserts of cDNA cloneswereused forhybridization.ForSouthernblottingDNAwasextracted bylysisofpurifiednucleiat65°Cinasolutioncontaining0.2M EDTA, pH 8.4,and 2%Sarcosylandpurified bycentrifugationinadensitygradientof CsCl with ethidiumbromide(Noegeletal., 1985).EcoRI andHindIIIfragmentswere

separatedon0.7% agarosegelsinTris-phosphate buffer, pH7.8(Maniatiset

al., 1982).

Forhybridization,filterswereincubated with nick-translatedprobesfor 18-20h at37°C in 2 x SSC,50% formaldehyde,4 mMEDTA, 1% Sarcosyl,0.1%

SDS,4 xDenhardt's and 0.12Mphosphate buffer, pH6.8(Mehdy^etal.,1983).

The filterswerewashed in thesamesolution for1hat37°Candautoradiographed

onKodakXARfilm.

Acknowledgements

WearegratefultoDrsRichard Kessin and Marie-LiseLacombeforkindlypro- viding uswith the Xgtll-cDNAlibrary. We thank also Barbara Fichtner and DanielaRiegerformonoclonalantibodyproductionandKlaus Weber forcon-

struction of thesingle-cell depositionsystem.

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Receivedon21 October 1985