Vol. 81, No. 3, 1978

BtOCHEMlCAL

AND BIUPHYSICAL RESEARCti COMMUNICATIONS

Pages 814-821

Aprit 14, 1978

THE EFFECTON2,k-DINITROPHENOLON DICTYOSTELIUMDISCOIDEUMOSCILLATIONS

J. Geller end M. Brenner Harvard Biological

Laboratories

Cambridge, Massachusetts 02138 Received

February

27,1978

During aggregation the cellular slime mold Dictyostelium discoideum emits pulses of CAMPabout every 5 minutes. Onlv a small fraction of the aagregating cells produces the pulses autonomously, while most cells synthesize and release the nucleotide in a chain-reaction response to the autonomous signals (1). Wereport here that 2,b-dinitrophenol, KCN, and caffeine all inhibit the autonomousCAMPoscillations but do not interfere with the triggered response. Because of this, and other data (21, we question current models of the oscillatory synthesis of CAMP.

INTRODUCTION Oscillatory zation,

phenomenaare observed at all

levels of biological

orgsni-

from the social to the molecular, with periods ranging from years to

seconds (3).

One of the simplest eukaryotes,

plays oscillatory

Dictyostelium

discoideum, dis-

phenomenaduring the aggregation phase of its development.

Time-lapse cinematography of amebaeaggregating on a surface reveals that chemotaxis towards the aggregation center is not continuous, but occurs in pulses of movement. These pulses of inward movement spread as slow waves out from the center, utes. trolled

new

waves being produced with a period of about 5 min-

This process is now understood to be initiated synthesis and release of the attractant

cells at the center of the territory

by the internally-con-

, CAMP, from one or a few

(autonomous cells).

These pulses are

detected by adjacent cells which respond by chemotaxing towards the source, and also by emitting their

own pulse of CAMP(relay-competent

0006-291X/78/0813-0814$01,00/Q Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any formreserved.

814

cells).

Hence,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 3, 1978

two processes

are involved

autonomous cells,

and signal

An important observation

transmission

technical

of Gerisch

in scattered-light tion-phase

in the generation

advance in studying

intensity

occurred

amebse.

are accompanied by oscillations trstion

of CAMP (5),

it

controls

signal

nism that

What this speculation.

is identical tal with

studies

mechanism. press cells.

was the

oscillations

suspensions

of aggrega-

Since these light-scattering in the extracellular

generation

in cell

production

in details,

oscillations

and intracellular

layers

for autonomous and of the oscillator

concen-

point

Here we show that

autonomous oscillations

a limit-cycle

2,b-dinitrophenol,

relay

a subject

affecting

does not utilize

Although

that experimen-

inhibitors,

in the oscillator

KCN, and caffeine the relay

has

differing

oscillator

of metabolic elements

for

of cells

We have initiated

the effects

to controlling

without

remains

and Segel (7).

cells.

relaying

mecha-

on agar surfaces.

of CAMP by a population

by testing

they will

that

level

both models utilize

by

(1).

is assumed that they are due to the same basic

The periodic

This suggests

to seven-minute

in illuminated

mechanism is at the molecular

the hope that

cells

the CAMP oscillations

five-

been modeled by Cohen (6) and by Goldbeter substantially

a triggering

by relay-competent

and Hess (4) that

2. discoideum

of a CAMP wave,

can sup-

competence of

the s&me basic

oscillator

as autonomous CAMI?production.

METHODS

Cells of k discoideum strain A3 (8) were grown in HL-5 medium, harvested and washed twice with cold distilled water as previously described (9). Cells were resuspended at a density of 2xlOT/ml in 16 mM MESSKOH [2-(N-morpholinoj-ethane sulphonic acid] pH 6.0; or for pH measurements, in unbuffered 16 mM KCl. Because the cells alkalinize the unbuffered medium at a constant rate, a constant flow of HCl was used to maintain the extra-cellular pH between 6.0 ma 6.2. Oscillations in light-scattering were observed by placing an aerated cell suspension in the light beam of a Zeiss PMQII spectrophotometer and recording transmitted light intensity at 540 nm. For CAMP measurements samples of cell suspension were rapidly transferred to equal volumes of 10% (w/v) trichloroacetic acid end vortexed. CAMPwas purified ma assayed as described previously (9) except that the alumina columns were omitted. Levels of CAMPobtained following an exogenous pulse were c rrected for CAMP remaining from that pulse. This was done by including 4x10e cpm [3H3cAMP(40

815

Vol. 81, No. 3,1978

BIOCHEMICAL AND BlOPHYSlCAl RESEARCH COMMUNICATIONS

11

IbE&O.O2J

t

t

I

IO

Fig.

20

30 Timr (min)

40

50

1. CAMP and light-scattering oscillations. A) Cells were prepared and CAMP(0) measured as described in Methods. B) Arrows indicate when 1 mMCAMPwas added to the cell suspension to a final concentration of 1 uM. AE refers to absorbance; the arrow indicates increasing absorbance.

Ci/mmol, New England Nuclear) per ml cell suspension and determining by chropH was monitored matography (9) the amount remaining as CAMP. Extracellular with a Beckmanglass pH probe.

RESULTSANDDISCUSSION

Typical

light-scattering

and CAMPoscillations

suspension of 2. discoideum are shown in Fig. IA.

for an illuminated Also shown is the effect

of pulsing the suspension with CAMP(Fig. 1B). The light-scattering sity cells

decreases

oscillations

light

inten-

within 15 s of adding 33 PM DNRto a suspension of oscillating

(Fig. 2).

slowly levels off.

This decrease in intensity Similar effects

15 @4; below this concentration

However, addition

is followed by an increase which

occur for

no effects

Total CAMPlevels stop oscillating =I.

cease and the transmitted

DIP concentrations

are observed.

after

addition

of 33 PM DNP (Fig.

of 1 UMCAMPto DNP-treated cells elicits

816

greater than

changes in

8lOCHEMlCAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

250

Time

Fig. 2. A)

B)

(mid

Effect of DNPon light-scattering and CAMPoscillations and on relay competence. Cells were prepared and CAMP(0) measured a8 described in Methods. The first arrow indicates when 100 mMDNPwas added to the suspension to a final concentiration of 33 uM. The three subsequent arrows, labeled Kl, K2, K3, indicate additions of 1 M KOHto final increments of 3 mMeach. Numbersunder these arrows indicate extracellular pH. The arrows over the trace of restored oscillations indicate where CAMPpeaks would appear were phase preserved. Cells were prepared and CAMP(0) measured as described in Methods. DNPwas added as above. To test for relay competence, CAMPwas added to a final concentration of 1 PM, where indicated, from a 1 mMstock solution. The first time point was taken 15 s after addition of CAMP. AE refers to absorbance; the arrow indicates increasing absorbance.

light-scattering

(Fig.

2B) which are identical

is added to a suspension of untreated cells reproducibly

to those recorded when CAMP

(Fig. 1B).

obtained upon subsequent additions of CAMP. In someexperi-

ments, in which the cells were several hours older, scattering

This response can be

oscillations

The level of total

returned after

two to three additions of 1 fl

endogenousCA!@after

with 1 fl CAMPis indicated

spontaneous light-

stimulation

CAMP.

of DNP-treated cells

in Fig. 2B. As can be seen, addition of exogen-

ous CAMPstimulates a very rapid synthesis of CAMPby the cells.

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Vol. 81, No. 3, 1978

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AND BlOPHYSlCAL

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Time

Fig. 3.

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RESEARCH COMMUNICATIONS

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60

I

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Effect of DNF on extracellular pH oscillations. Cells were prepared as described in Methods for pH measurements. DNPwas added as in Fig. 2 where indicated. After DNPaddition the HCl source was removed to allow the cells to alkali&se the suspension. Numbersnext to the pH trace indicate the extracellular pH at those points. The arrows over the trace of restored oscillations indicate where CAMPpeaks would appear were phase preserved. AE refers to absorbance; the arrow indicates increasing absorbance. The extracellular pH increases in the direction indicated.

In unbuffered cell suspensions we have observed spontaneous oscillations in extracellular

pH that are in phase with CAM?oscillations

Observation of these pH oscilla-

reported by Nanjundiah and Malchow (lo)]. tions is obscured unless the alkalinization by constant addition of HCl. light-scattering,

flow of HCl is stopped after

DNPaddition,

to resume (Fig. 3).

creased extracellular

the alkalinisation allowing light

The reversal

of

scattering

of DNF inhibition

are again inhibited

If the

and pH

by the incoeffi-

If the pH is restored to 6.0 all

(data not shown). 818

and

the medium

pH is probably due to a change in the partition

cient of DN'Pacross the cell membrane(11). oscillations

in CAMPconcentration

are eliminated by DNP (Fig. 3).

by the cells reverses the DNP inhibition, oscillations

of the mediumis counterbalanced

Like the oscillations

the pH oscillations

[also

BfOCHEMtCAt

Vol. 81, No. 3,1978

The data in Fig. 2 indicate

AND BtOPHYStCAL RESEARCH, COMMUNtCATtONS

that the cellular

CAMPsynthesizing

(adenylate cyclase, ATP) and CA?@receptors are functional tion.

The cessation of CAMPoscillations

be due to an effect

efflux

intact,

it

remains intact,

of CAMPis disrupted.

argue against the latter

cells.

possibility.

is destroyed while

but its driving

link to

Data presented in Figs. 2 and 3

Were the basal oscillator

to remain

should maintain its phase in the presence of DNP. This prediction

can be tested as the effects increased to about 7.0. selves alkalinize

of DNPare reversed if the extracellular

In unbuffered suspensions (Fig. 3) the cells

the medium; in buffered

suspensions it

addition of KOH (Fig. 2A).

As shown in these figures,

stored oscillations

from that existing

trol

One possibility

in the autonomous cell itself

another is that the basal oscillator

DNPaddi-

DNPaddition must therefore

on the autonomously oscillating

is that the basal oscillator

the periodic

after

after

system

differs

prior

pH is them-

can be achieved by

the phase of the reto DNPaddition.

Con-

experiments showedaddition of KOHalone does not cause phase-shifting.

It is still

possible that the basal oscillator

remains intact,

but that DNP

changes its period in addition to uncoupling it from CAMPproduction. type of model The initial

is difficult

to test experimentally.

signal leading to activation

fers in autonomouscells from the basal oscillator, CAMPto the external

This

and relaying

cells;

of the adenylate cyclase difin the former the signal comes

whereas in the latter

it

comesfrom binding of

cell membrane. However, it is unknown if the two sig-

nals subsequently share

common

steps in their

disrupts autonomous CAMPoscillations

The fact that DNP

but not CAMPrelay suggests that at

least somefacets of the basal oscillator

in autonomous cells are not shared

by the relay system in relay competent cells. models in which a unified

processing.

This finding

argues against

mechanismfor autonomousoscillations

and relay is

proposed (7). In conjunction with studies to be published elsewhere (2), we extend our reSerVatiOnS

concerning the unified

oscillator

819

models to question whether

Vol. 8 1, No. 3, 1978

Fig. 4.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Effect of caffeine on light-scattering and CAMPoscillations and on relay competence. Cells were prepared and CAMP(0) measured as described in Methods. The first arrow indicate5 when 100 mMcaffeine was added to the suspension to a final concentration of 5 mM. The second arrow inaicatee when relay competence was tested by addition of CAMPas in Fig. 2B. AE refers to absorbance; the arrow indicate5 increasing absorbance.

production of CAMPby relaying

cells

involves s,n oscillator

at all.

The

models of both Goldbeter and Segel (7) and of Cohen (6) are predicated on oscillations

of control metabolites that occur with the s&meperiod, but dif-

ferent phase, as CAMP. Wehave found no evidence for such oscillation5 metabolites during light-scattering that metabolite oscillations

oscillations

(2).

We

suggest, therefore,

occur only in autonomouscells.

about one cell in lo4 is autonomous (l),

oscillations

of

Because only

in their

metabolites

would not be measurable against the background of non-autonomous cells. At this point we do not know how DNPacts to inhibit Other compounds,which are thought to have different similar pattern5 of inhibition

but has no effect

coideum caffeine

is not an inhibitor

it alter5 the intracellular cells

(13).

metabolic effects,

as DNP. Like DNP, caffeine

ous oscillations,

on signal relay

inhibit5

(Fig. 4).

give

spontane-

In g. $&-

of CAMPphosphodiesterase (12); possibly

distribution

Although its effect5

oscillations.

of Cay a5 has been found for muscle

on CAMPlevels were not determined, oscillations

50

PM

KCNhas the s&meeffect

on light-scattering

as DNPand caffeine;

autonomousoscillations

are suppressed, but the response to exogenous CAMPis

normal (data not shown, but see Fig. 2 or 4 for similar traces).

820

Vol. 81, No. 3, 1978

8lOCHEMlCAL AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 2: 7. 8. 9. 10. 11. 12. 13.

Raman, R. K., Htlehimoto, Y., Cohen, M. H., and Robertson, A. (1976) J. Cell Sci. 2l, 243 Geller, J., and Brenner, M., manuscript in preparation Goldbeter, A., and Caplan, S. R. (1976) Ann. Rev. Biophys. Bioeng. 2, 449 Gerisch, G., and Hess, B. (1974) Proc. Natl. Acad. Sci. USA 1, 2118 5 Gerisch, G., and Wick, U. (1975) Bioch. Biophys. Res. Comm. 2, 364 Cohen, M, (1977) J. Theoret. Biol. 6 , 57 Goldbeter, A., and Segel, L. A, (19773 Proc. Natl. Acad. Sci. USA &, 1543 Loomis, W. F., Jr. (1971) Exp. Cell Res. 64, 484 Brenner, M. (1977) J. Biol. Chem. 2 2, 4073 NeJ#.uidiah, v. ) and Malchox, D. (1973-T Hoppe Seyler's 2. Physiol. Chem. m, 273 Christensen, H. (1975) Biological Transport, 2nd ed., pp. 69-70, W. A. Benjamin, Inc., Reading, Ma. Chang, Y. (1968) Science 161, 57 Endo, M. (1977) Phys. Rev. 51, 71

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