J. Cell Sci. 10, 249-265 (1972) 249

Printed in Great Britain

CELL ELECTROPHORESIS OF THE CELLULARSLIME MOULD DICTYOSTELIUM DISCOIDEUM

II. RELEVANCE OF THE CHANGES IN CELL SURFACECHARGE DENSITY TO CELL AGGREGATION ANDMORPHOGENESIS

K.-C. LEESub-Department of Cliemical Microbiology, Department of Biochemistry,University of Cambridge, Cambridge, England

SUMMARY

The reduction in the cell surface charge density of Dictyostelium discoideum during differentia-tion has been studied by the technique of cell electrophoresis. It was abolished under conditionsin which cell aggregation was inhibited (e.g. low temperature, the presence of actinomycin Dor cycloheximide). In the presence of nutrients incapable of supporting growth, cell aggregationoccurred without a reduction in surface charge density. Cell adhesion in these aggregates wasimpaired, and a reduction in surface charge density appeared to be necessary for furtherdevelopment.

Brief treatment of exponential phase and aggregating cells with agents which disaggregateslugs failed to alter their electrophoretic mobilities. Low concentrations of magnesium chloridecaused extensive agglutination, especially in aggregating cell suspensions, but little change intheir electrophoretic mobilities. Magnesium chloride could agglutinate cells by association withcell surface components undetectable by cell electrophoresis. This, together with immuno-logical evidence from other workers, supports the possibility of involvement of specific surfacemacromolecules in cellular slime mould aggregation. It was concluded that changes in surfacecharge density, though important for cell adhesion and morphogenesis, cannot account for allaspects of cell interactions in D. discoideum.

INTRODUCTION

The electrophoretic mobility (and hence the surface charge density) of Dictyosteliumdiscoideum amoebae decreases progressively as they approach cell aggregation (Garrod& Gingell, 1970). This phenomenon is probably due to a decrease in the density of cellsurface carboxyl groups, and an increase in that of amino groups (Lee, 1972). Thereduction in surface charge density of the cells on incubation in a non-nutrientmedium can be roughly correlated with an increase in their agglutinability by simplesalts (Born & Garrod, 1968), and the increased resistance of cell clumps to mechanicaldispersion. It is not known if a reduction in surface charge density is obligatory for cellaggregation, or is merely an unrelated phenomenon associated with starvation. Oneapproach to this problem is to investigate the effect of various agents which inhibit celldifferentiation or disaggregate pseudoplasmodia (slugs) on the electrophoretic mobilityof the cells.

Factors inhibiting cell aggregation and morphogenesis in D. discoideum include lowtemperature (Garrod & Gingell, 1970), actinomycin D, cycloheximide and nutrients.

250 K.-C. Lee

Actinomycin D is a recognized inhibitor of RNA synthesis, and was used to inhibitslime mould morphogenesis (Pannbacker & Wright, 1966; Wright & Pannbacker,1967; Sussman, Loomis, Ashworth & Sussman, 1967; Hirschberg, Ceccarini, Osnos& Carchman, 1968), and as a specific inhibitor of RNA synthesis in studies of develop-mentally linked enzymes (Sussman, 19666; Loomis, 1970; Telser & Sussman, 1971;Newell, Longlands & Sussman, 1971). Cycloheximide (actidione), a well knowninhibitor of protein synthesis in eukaryotic cells, was used to inhibit protein synthesisand formation of developmentally linked enzymes in D. discoideum (Sussman, 1965,and the papers on actinomycin D).

The agents which disaggregate slugs and aggregates include EDTA and proteases.Sussman & Sussman (i960) used trypsin to disperse aggregates of a 'fruity' mutant ofD. discoideum (Strain Fty-i) for cell counting, but Whitfield (1964) could not repeatthis for wild type D. discoideum, and found that only papain was effective under alimited range of conditions. Papain was also used by Yanagida & Noda (1967).Takeuchi & Yabuno (1970) reported that D. discoideum slugs could be disaggregatedby a variety of proteases in the presence of 2,3-dimercapto-propan-i-ol and simplesalts.

EDTA has been used for disaggregating slugs or aggregates (Dehaan, 1959;Gerisch, 1961; Shaffer, 1962; Whitfield, 1964; Takeuchi & Yabuno, 1970), but theresults of different authors are conflicting and may reflect the experimental conditionsused. EDTA prevents clumping of D. discoideum cells which have been incubated for2 h, but does not appreciably alter their zeta potential (Gingell & Garrod, 1969;Gingell, Garrod & Palmer, 1969). Theae authors concluded that EDTA does notinhibit cell aggregation simply by increasing the net electrostatic repulsive forcebetween cells through chelation of divalent cations. However, unlike other electro-phoretic studies on divalent cation binding (Weiss, 1968; Seaman, Vassar & Kendall,1969; Patinkin, Schlessinger & Doljanski, 1970; Patinkin, Zaritsky & Doljanski,1970), magnesium ions were not present in the electrophoresis buffer of control cells,and it was not known whether their presence would lower the cell surface chargedensity. Another criticism is that the cells used for the experiments had been incubatedfor only 2 h, and the clumping of these cells is not the same as cell aggregation 4-5 hlater, even though EDTA may inhibit both by the same mechanism.

The object of the work described in this paper was to establish the relevance of thesurface charge density changes to cellular slime mould aggregation and morphogenesisby investigating changes in electrophoretic mobility under conditions unfavourable tocell differentiation or the existence of aggregates.

MATERIALS AND METHODS

Growth and differentiation of Dictyostelium discoideum

The methods for growth of D. discoideum Ax-2 in axenic culture and differentiation onMillipore niters, agar or in non-nutrient liquid culture have been described (Lee, 1972).

In some experiments, differentiation was conducted statically in liquid medium. Cells wereharvested during the exponential phase of growth, washed twice at o °C, and resuspended inmedium M (10 mM KC1, 5 mM MgClj, 10 irw sodium phosphate, pH 6-o). The cell suspension

Slime mould cell electrophoresis. II 251

was pipetted into 6- or g-cm plastic Petri dishes so that each dish contained io7 cells and 3 mlmedium or 3 x 1 0 ' cells and 10 ml medium respectively. The cells soon settled to the bottom,and were not resuspended by slight movement of fluid in the dish. The presence of magnesiumions was essential for firm attachment of the amoebae to the bottom of the dish. The disheswere incubated statically in the dark at 23 °C. Under these conditions, the formation of streamsand aggregation centres was detectable after 9—10 h, and aggregation completed by 16—20 h.Sussman's medium (Sussman, 1966a) can be used instead of medium M, but differentiation isslower.

The following additions were made to medium M in the Petri dishes to prevent cell aggre-gation: (1) 1 6 2 % (w/v) glucose. (2) An amino acid mixture of the same composition as caseinhydrolysate. Final concentrations in Petri dishes (TTIM) Asp, 2-43; Thr, 0-94; Ser, 180; Glu,4-96; Pro, 2-82; Gly, 1-17; Ala, 1-37; Val, 0 9 3 ; Met, 0 4 3 ; He, 0 4 8 ; Leu, 145; Tyr, 0-26;Phe, 0 4 8 ; Lys, 148; His, 0-30; Arg, 039. Total: 2i '7mM. (3) A vitamin mixture. Finalconcentrations in Petri dishes (/ig/ml); biotin, 0-02; calcium pantothenate, and folic acid, 6 0 ;pyridoxine, nicotinic acid, inositol, thiamine and ^-amino-benzoic acid, 660.

Cell electrophoresis

This was performed as described elsewhere (Lee, 1972).

MaterialsCrystalline trypsin (EC 3.4.4.4) and papain (EC 3.4.4.10) were purchased from Worthing-

ton Biochemical Corp., Freehold, N. J.; pronase (B grade) from Calbiochem Ltd.; snail gutdigestive juice and cycloheximide from Koch Light Laboratories Ltd.; 2,3-dimercapto-propan-i-ol (British Anti-Lewisite, BAL) from British Drug Houses; and actinomycin D was a giftfrom Merck, Sharp and Dohme Ltd., Hoddesdon, Herts.

RESULTS

Effect of agents which disaggregate slugs on the electrophoretic mobility

EDTA. EDTA has been used by a number of workers to disperse aggregates andslugs of D. discoideum, and its effect on the electrophoretic mobility of preaggregationcells was studied by Gingell & Garrod (1969) and Yabuno (1970).

Incubation of exponential phase cells, aggregating cells and aggregation-competentcells with 10 raM EDTA for 30 min had only marginal effects on their electrophoreticmobilities (Table 1). Although EDTA dispersed aggregates under the conditionsemployed, the cell suspensions obtained formed clumps of 10-20 cells on standing in10 mM sodium phosphate (pH 6-8) at room temperature for 20 min, even in the pre-sence of 1 mM EDTA. Under the same conditions, exponential phase cell suspensionsremained essentially single-celled. These findings are in aggreement with those ofGerisch (1961, 1968) that aggregation-competent cells formed clumps in the presenceof EDTA. Aggregating cells dispersed with EDTA rapidly recovered and completedtheir development (Takeuchi & Yabuno, 1970). Although EDTA causes loss of surfacemacromolecules from bacteria (Leive, Shoulin & Mergenhagen, 1968), no evidencewas obtained for removal of specific adhesive substances from the surface of slimemould cells by a 30-min treatment with EDTA. However, when cells were incubatedon Millipore filters in the presence of 10 mM EDTA, cell aggregation and the fall inelectrophoretic mobility were inhibited. After incubating for 10 h in the presence ofEDTA, only 10% of the cells completed their development on removal of EDTA. It

252 K.-C. Lee

Table i. Effect of EDTA treatment on the electrophoreticmobility of D. discoideum

Electrophoretic mobility( - / i m r 1 V"1 cm ± S.E.M.)

Cells Control Treated

Student's t-testprobabilities,

control v. treated

Exponential phase 1-42 ±0-02 (10)

Aggregation- 1-15 ±0-02 (17)competent (10-5 hin medium M)

Aggregating cells 083 ±o-oi (11)(12 h on Milliporefilters)

I-37±O-OI (10)

1-22 ±O-O2 (2l)

o-i < P < 05

OOI < P < 0-05

I±o-O2 (10) o-oi < P < 0-05

Cells harvested during the exponential phase of growth were incubated on Millipore filtersor in suspension in medium M for 12 and 10-5 h respectively. They were harvested, washedand resuspended in 0145 M NaCl, iomM Tris-EDTA, iomMTris-HCl (pH 7-5) at io7 cells/ml.The suspension was shaken for 30 min at 23 °C, 150 cycles per min in an orbital incubator.Electrophoresis was carried out in iomM sodium phosphate (pH 6-8, / = 0-02 g ion I."1) aftertreatment. Student's t test probabilities (control v. treated) are shown. The numbers ofobservations made are in parentheses.

1-4

S 1-3

5 >

1 2 -]

1-1

1 0

09

08

J_ I|10- 5X10-

1 10"1

MMgCla2x10" 5x10-

Fig. 1. Effect of magnesium ions on the electrophoretic mobility of D. discoideum.Exponential phase cells and aggregating cells (after 10 h on agar) were washed onceand resuspended in sodium phosphate (pH 6-8) containing different concentrations ofmagnesium chloride and the electrophoretic mobilities were determined. The ionicstrength of the solution was maintained at 0-02 g ion I."1. 9 , exponential phase cells;O, aggregating cells. The histograms show the electrophoretic mobilities of exponentialphase cells (hatched) and aggregating cells (unhatched) in 1 mM Na, EDTA, 7 mMsodium phosphate (pH 6-95-7-00, / = 0-02 g ion I."1).

Slime mould cell electrophoresis. II 253

was probable that prolonged treatment resulted in irreversible interference with cellmetabolism.

An alternative explanation for the dispersal of aggregates by EDTA is that itchelates divalent cations bound to the cell surface, and increases the zeta potential. Theeffect of a divalent cation (Mg2+) on the electrophoretic mobilities of exponential phaseand aggregating cells was studied (Fig. 1). Magnesium ions had little effect on theelectrophoretic mobilities of both cell preparations at concentrations up to 2 rriM, butproduced extensive clumping, especially in suspensions of aggregating cells. At 5 mMMgCl2) the electrophoretic mobilities of both cell preparations were reduced by about20%. It was not possible to determine electrophoretic mobilities at greater magnesiumion concentrations due to massive cell clumping. The presence of EDTA (1 mM) inthe electrophoresis buffer caused a significant (P < o-ooi) but small increase in theelectrophoretic mobility of exponential phase cells, but not in that of aggregating cells

(P > o-5).Enzymes. Dictyostelium discoideum pseudoplasmodia are effectively disaggregated by

papain and pronase in the presence of 2,3-dimercapto-propan-i-ol (BAL) (Takeuchi &Yabuno, 1970), and the effect of these and other enzymes on the electrophoreticmobility of exponential phase and aggregating cells was studied (Table 2). It is clearthat treatment with enzymes, irrespective of their ability to disaggregate slugs on agar,produced only marginal effects on the electrophoretic mobility of both cell preparations,even though some of the changes were statistically significant (P < 0-05).

Factors affecting cell aggregation and development

Temperature. Growth and development of D. discoideum did not occur at 4 °C, andat this temperature there was no fall in electrophoretic mobility when incubated in anon-nutrient medium (Fig. 2). This finding is in agreement with the results of Garrod& Gingell (1970) for pre-aggregation cells.

Inhibitors of nucleic acid and protein synthesis. The 2 inhibitors used most extensivelyin studying cellular slime mould development are actinomycin D and cycloheximide,and the effects of these on the changes in electrophoretic mobility which occur duringthe early stages of differentiation were investigated. Cells harvested during the ex-ponential phase of growth were incubated on Millipore filters, arrd after various timeintervals the filters were transferred to support pads containing actinomycin D. Cellswere harvested at intervals, and their electrophoretic mobilities determined (Fig. 3).Addition of actinomycin D at any time up to 5 h inhibited cell aggregation and abolishedany further decrease in electrophoretic mobility. There was a rise in electrophoreticmobility towards the initial value for cells which had been incubated for 4 and 5 hbefore addition of the inhibitor. Addition of actinomycin D at 6-5 h did not prevent thefall in electrophoretic mobility or inhibit cell aggregation. This failure to inhibitaggregation has been attributed by Sussman et al. (1967) to the presence of mRNAsynthesized before addition of the inhibitor.

In contrast to actinomycin D, cycloheximide inhibits differentiation immediately,and its addition to cells at o and 6-5 h immediately prevented any further fall inelectrophoretic mobility, and inhibited cell aggregation (Fig. 4).

Tab

le 2.

Efle

ct o

f en

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ic t

reat

men

t on t

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ty o

f D

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Ele

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ph

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mo

bil

ity

(-p

m

s-I

V-'

cm f s

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.)

A

r >

Enz

yme

and

E

ffec

tive

ness

in

Ex

po

nen

tial

pha

se c

ells

A

gg

reg

atin

g c

ells

d

ura

tio

n o

f di

sper

sing

slu

gs

Ele

ctro

phor

esis

I

A

> f

A

>

trea

tmen

t o

n a

gar

buff

er

Co

ntr

ol

Tre

ated

C

on

tro

l T

reat

ed

Sna

il g

ut

juic

e I

h -

10

rn

~

sod

ium

-

Pro

nase

/ 5

10 r

n~

so

diu

m

mer

capt

oeth

anol

p

ho

sph

ate

(PH

6.8

) P

ron

ase/

BA

L

30 r

nin

f

I o

mM

so

diu

m

I .4

6 5 0

.02

I .4

0 f 0

.02

I .0

2 + 0

.01

r .06

f 0

.01

ph

osp

hat

e (1

2)

(1 I

) (1

2)

(1 2)

(P

H 6

.8)

0.0

1 <

P <

0.0

5 0.

05 <

P <

0.1

Pro

nase

IBA

L

+ 0.

145

M N

aCl

-

-

0.60

f 0

.01

0.61

f 0

.01

30 m

in

3 x

10

4

M N

aH

C0

3

(9)

(9)

(pH

7.2

) P

> 0

.5

Exp

onen

tial

ph

ase

cell

s an

d a

ggre

gati

ng c

ells

(af

ter

ro h

inc

ubat

ion

on

2 %

aga

r) w

ere

har

ves

ted

an

d i

ncu

bat

ed a

t a

cell

den

sity

of

10' ce

lls/

ml

at 2

3 O

C,

150

cycl

es p

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in a

n o

rbit

al i

ncub

ator

in

the

foll

owin

g en

zym

e so

luti

on

s: (

i) T

ryp

sin

: 0

.1 r

ng/r

nl i

n 1

0 m

M

CaC

I,,

50 m

~ T

ris-

HC

1 (

pH

8.0

); (

ii)

Sna

il g

ut

dige

stiv

e ju

ice:

2 %

(vl

v) i

n 10 m

M s

od

ium

ph

osp

hat

e (p

H 7

.0);

(ii

i) P

apai

n:

0.3

mg

/ml

in z

o m

~ N

aCI,

20 m

~ K

C1,

2.5

m~

BA

L (

2,3

dir

nerc

apto

-pro

pan-

1-01

) in

10 rn

M s

od

ium

ph

osp

hat

e (p

H 6

.0);

(iv

) P

ron

ase:

I m

g/r

nl

in

20 m

~ N

aCI,

20 m

~ K

CI,

2.5

mM

BA

L o

r m

erca

pto

eth

ano

l an

d 30

mM

Tri

s-H

C1

(p

H 7

.4).

Aft

er t

he

peri

od o

f ti

me

sho

wn

in t

he

tab

le,

the

cell

s w

ere

harv

este

d, w

ashe

d an

d re

susp

end

ed f

or e

lect

roph

oret

ic m

easu

rem

ents

. T

he

nu

mb

ers

of o

bse

rvat

ion

s m

ade

are

in p

aren

- th

eses

. S

tud

ent's

t-t

est

prob

abil

itie

s (c

ontr

ol v

. tr

eate

d)

are

show

n.

Co

ntr

ols

wer

e in

cub

ated

un

der

ide

ntic

al c

on

dit

ion

s b

ut

wit

ho

ut

the

enzy

mes

. T

he

exp

erim

ents

usi

ng

dif

fere

nt e

nzym

es w

ere

perf

orm

ed o

n s

epar

ate

occa

sion

s. T

his

an

d t

he

dif

fere

nt

incu

bat

ion

co

nd

itio

ns

coul

d ex

plai

n th

e di

ffer

ence

s in

th

e co

ntro

ls.

'Ag

gre

gat

ion

-co

mp

eten

t' ce

lls

incu

bat

ed i

n su

spen

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10 m

M s

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ph

osp

hat

e (p

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.8)

for

12

h w

ere

use

d.

Slime mould cell electrophoresis. II 255

i-5 r

Fig. 2. Effect of incubation at low temperature on the electrophoretic mobility ofD. discoideum. Cells harvested during the exponential phase of growth were incubatedin liquid culture at a cell density of io'/ml in 10 mM sodium phosphate (pH 6-8) in anorbital incubator at 23 °C or 4 °C. Samples were taken at various times, and theirelectrophoretic mobilities determined in 10 mM sodium phosphate (pH 6-8, / = 0-02 gion I.-1). • . cells incubated at 23 °C; Q, 4 °C.

Fig. 3. Effect of actinomycin D on the electrophoretic mobility of differentiating cellsof D. discoideum. Cells harvested during the exponential phase of growth were incu-bated on Millipore niters. At o, 4, 5, and 6-5 h, filters were transferred to supportpads containing 2-5 ml 10 mM sodium phosphate (pH 6-8) with 125 /ig/ml actino-mycin D, and incubation was continued. Cells from 2 or more filters were harvestedat intervals, and their electrophoretic mobilities determined in 10 mM sodiumphosphate (pH 6-8, / = 0-02 g ion I."1). The arrow indicates the onset of aggregationin the control. # , control; O» treated with actinomycin D.

256 K.-C. Lee

Effect of nutrients. Dictyostelium discoideum did not develop aggregation competencein the growth medium, and on continued incubation into the stationary phase ofgrowth, lost the ability to differentiate. Watts & Ashworth (1970) reported thatD. discoideum cells would grow and form colonies on agar containing the growthmedium, but rarely formed fruiting bodies. It was decided to look for simple mixturesof nutrients which would inhibit aggregation.

1 5 1-

Fig. 4. Effect of cycloheximide on the electrophoretic mobility of differentiating cellsof D. discoideum. The experimental conditions were the same as in the legend to Fig. 3,except that the cells were transferred to support pads containing 400 /

Slime mould cell electrophoresis. I1

a 'I: A

3 S $ m .s - " a C A S g a !? 3 c-3 2 .s

C E L I 0

258 K.-C. Lee

without a fall in the electrophoretic mobility of the cells, the aggregates produced(Fig. n ) appeared less firm and compact than those produced in the control (Fig. 7),and were readily dispersed to form a suspension of single cells and some small clumps.The control aggregates required several washes in ice-cold buffer and vortex-mixingfor dispersal (Fig. 15). It is clear that in the presence of glucose (and amino acids plusvitamins), cell aggregation occurred in the absence of a fall in electrophoretic mobility,but cell adhesiveness was reduced.

10 11 12 13 14 15 16 17

Fig. 5. Effect of incubation with glucose on the electrophoretic mobility of differentiat-ing cells of D. discoideum. Cells harvested during the exponential phase of growth wereincubated on Millipore filters with or without 1-62 % (w/v) glucose. Cells from two ormore filters were harvested at various times, and their electrophoretic mobilities deter-mined in 10 mM sodium phosphate (pH 6-8, / = 0-02 g ion I."1). The arrows indicatethe times at which cell aggregation occurred. O> incubated with glucose; # , withoutglucose.

Morphogenesis of D. discoideum after cell aggregation did not occur under water,and incubation on Millipore filters was used to study further development of aggregatesformed in the presence of glucose. The majority of these aggregates formed slugs andfruiting bodies (Fig. 11-14). The variation in electrophoretic mobility of D. discoideumincubated on Millipore filters with or without glucose is shown in Fig. 5. There wasvirtually no change in the electrophoretic mobility of cells incubated in the presenceof glucose during the first 12 h, even though cell aggregation was discernible after 8 h.The aggregates dispersed fairly easily on washing off the filters, and the distribution ofelectrophoretic mobilities was unimodal at all times up to 12 h. After 14-5 h incuba-tion, cells with lower electrophoretic mobilities were evident, and after 16-5 h, thedistribution of electrophoretic mobilities was distinctly bimodal (Fig. 6). Only thelower mean value of electrophoretic mobilities is plotted in Fig. 5 for the i6-5-h timepoint. No evidence of a bimodal distribution was found for cells incubated in theabsence of glucose (Lee, 1972).

Slime mould cell electrophoresis. II 259

At 16-5 h (Fig. 6), high electrophoretic mobilities were associated almost exclusivelywith single cells which showed little or no pseudopodial activity in the electrophoresischamber. This behaviour was characteristic of non-aggregating cells. Low electro-phoretic mobilities were associated with single cells and many clumps which showedthe rapid pseudopodial activity characteristic of aggregating cells.

The appearance of cells with low electrophoretic mobilities after 12 h incubation

50

40

30

20

10

010 12 14 16

20

15

10

5

010 12 14 16 18 20 22 26

20

15

10

High Low

Ikl .—n10 12 14 16 18 20 22 24 26

Time, s, to traverse 10 graticule divisions (142 8 /im)

I | | | | |20 1-5 12 1 0 0 9 0 8

Electrophoretic mobility(— fim s"1 V"' cm)

Fig. 6. Distribution of electrophoretic mobilities of D. discoideum cells incubated withglucose. Cells incubated on Millipore filters in the presence of 1-62% (w/v) glucosewere harvested after various times for electrophoretic measurements. The electro-phoretic mobilities are represented by the time intervals taken by the cells to traverse10 graticule divisions (142-8 fim) when the applied voltage was 100 V. The times inhours, numbers of observations made and mean electrophoretic mobilities ( - / t mg- i v -

1 cmis.E.M.) were: A, 12, 55, 1-42 ±o-oi; B, 14-5, 123, 1-27 ±0-02; andc, 165,106 and high, 1-50 ±0-02, low i'O2 ±0-02.

17-2

260 K.-C. Lee

with glucose correlated approximately with the transition from the loose-lookingaggregates (Fig. n ) into more compact aggregates (Fig. 12) on Millipore niters. Avariable proportion, occasionally as high as 50%, of aggregates remained as shapelessclumps (Fig. 16) and failed to transform into slugs and fruiting bodies (Fig. 17).During migration on Millipore niters in the presence of glucose, the slugs sometimesleft behind large numbers of cells, some of which formed separate slugs, and some failedto develop further (Fig. 13). This could reflect an impairment of cell-cell adhesion.

The distributions of electrophoretic mobilities in Fig. 6 for 14-5 and 16-5 h areunreliable, as a proportion (up to 20%) of the cells harvested at these times remainedin large tight aggregates which were not dispersed by vortex-mixing. The proportionof cells with low electrophoretic mobilities is therefore an underestimate. Developmentafter aggregation is not possible if the cells are fully submerged, and the volume ofliquid in the support pads and the cell concentration on the Millipore niters werecarefully adjusted to give consistent results. Although cells incubated in the presenceof glucose exhibited small variations in different experiments in the times at which cellaggregation began and cells of low electrophoretic mobilities appeared, no appreciablefall in electrophoretic mobility was observed before cell aggregation occurred.

DISCUSSION

During the early stages of differentiation in D. discoideum there was a linear re-duction in electrophoretic mobility, and an increase in cell adhesiveness associated withincreased resistance of cell clumps to mechanical dispersion. These changes were notdissociated from cell aggregation by incubation at low temperature or in the presenceof actinomycin D or cycloheximide. When the cells were incubated in the presence ofglucose (or amino acids plus vitamins), there was no reduction in electrophoreticmobility before cell aggregation occurred, and cell adhesion in the resulting aggregateswas impaired. The transformation of these aggregates on Millipore filters into morecompact cell masses and eventually fruiting bodies was associated with the appearanceof cell clumps with increased resistance to mechanical dispersion and lower electro-phoretic mobilities. These changes may be due to a localized depletion of glucose inthe aggregates. It is likely that a decrease in the surface charge density, althoughunnecessary for cell aggregation, is essential for cell adhesion in aggregates and theirsubsequent development.

Since cell aggregation occurs in the absence of a reduction in surface negativecharge density, other surface components must be involved, for example, acrasinreceptors, species-specific recognition sites and intercellular adhesive substances.Cell aggregates and slugs are dispersed by treatment with proteolytic enzymes,but this treatment does not affect the electrophoretic mobility of the cells. It ispossible that the dispersal results from non-specific damage to the cell surface.This dispersal of aggregates without changes in surface charge density is consistentwith the hypothesis that the intercellular electrostatic repulsive force is not the onlydetermining factor in cellular slime mould aggregation.

The electrophoretic mobilities of exponential phase and aggregating cells were notchanged appreciably by treatment with EDTA or by the presence of EDTA in the

Slime mould cell electrophoresis. II 261

electrophoresis buffer. The presence of magnesium ions in the electrophoresis buffer atconcentrations which caused extensive clumping, especially of aggregating cells, didnot alter the electrophoretic mobilities of these cells. There is no evidence that EDTAinhibits cell clumping by chelating divalent cations. Magnesium ions could causeclumping by association with surface components undetectable by cell electrophoresis.

Yabuno (1970) reported that pronase or EDTA treatment resulted in an increase inthe electrophoretic mobility of D. discoideum (strain NC-4) which had been incubatedon agar for 3-5 h. It is not possible to assess the reliability of this result in the absence ofstatistical data, but the cells used had been grown on bacteria, and their electrophoreticmobilities would be expected to show considerable variation (Garrod & Gingell, 1970).Attempts to repeat this finding in D. discoideum (strain Ax-2) were not successful (Lee,unpublished).

There is evidence for the presence of intercellular binding substances in sponges(Humphreys, 1963, 1965, 1970), mammalian and avian cells (Moscona, 1962, 1968;Lilien, 1970). Complementary glycoproteins isolated from opposite mating types ofthe yeast, Hansenula wingei are believed to be involved in specific recognition duringmating (Crandall & Brock, 1968a, b). There is a large body of immunological evidencein the cellular slime moulds for antigenic differences between non-aggregating andaggregating cells (Sonneborn, Sussman & Levine, 1964; Gerisch, 1968; Beug,Gerisch, Kempff, Riedel & Kremer, 1970), between species (Gerisch, Malchow,Wilhelms & Liideritz, 1969) and between wild type cells and their aggregatelessmutants (Gregg & Trygstad, 1958; Gerisch & Beug, 1971). Although most of thesedifferences were demonstrated in crude preparations, and not always unequivocallyestablished to be on the cell surface, they may be involved in cell adhesion.

Cell aggregation in D. discoideum involves at least 2 processes: formation of aggre-gates by chemotaxis, and cell adhesion. The linear reduction in cell surface chargedensity during the early stages of differentiation is not specifically associated with theonset of cell aggregation and chemotaxis, but it can be correlated with a progressiveincrease in agglutinability and adhesiveness of the cells (Born & Garrod, 1968). Thischange in electrophoretic mobility is probably essential for strong cell adhesion inaggregates, and for their subsequent development. It is reasonable to suppose that theexistence of pseudoplasmodia of io6 cells or more and the complex morphogeneticmovements involved in migration and fruiting require strong cell-cell adhesion.However, changes in cell surface charge density cannot account for all aspects of cellrecognition, interaction and morphogenetic movements during aggregation, andsurface components undetectable by cell electrophoresis are probably involved.

I am indebted to Dr D. Kerridge for advice and encouragement during the course of thiswork and the preparation of the manuscript. Many thanks are due to colleagues in the Depart-ment of Biochemistry and elsewhere, especially Professor E. F. Gale and Dr K. McQuillen, fortheir interest and criticisms. I am grateful to the Wellcome Trust for a scholarship for trainingin research methods.

262 K.-C. Lee

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(Received 20 May 1971)

264 K.-C. Lee

Figs. 7-10. Differentiation of Dictyostelium discoideum on Millipore filters; after 10,16-5, 24, 36 h incubation respectively, x 3.Figs. 11-14. Differentiation of Dictyostelium discoideum on Millipore filters in the pre-sence of 1-62% (w/v) glucose; after 10, 16-5, 24, 36 h incubation respectively. Thearrows indicate cells that failed to fruit (Fig. 14). x 3.Fig. 15. Cells from one Millipore filter (5 x 10' cells) were washed off with 15 ml10 mM sodium phosphate (pH 6-8), and vortex-mixed for 5 s after 10 h incubationin the presence (A) or absence (B) of glucose. Actual size.Figs. 16, 17. Dictyostelium discoideum after 16-5- and 36-h incubations in the presenceof glucose, showing cells that did not develop after aggregation (arrows), x 3.

Slime mould cell ekctrophoresis. II 265