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Shuttle-Streaming: Synchronization with Heat Production in Slime Mold Author(s): Robert D. Allen, W. Reid Pitts,

Shuttle-Streaming: Synchronization with Heat Production in Slime Mold Author(s): Robert D. Allen, W. Reid Pitts, Jr., David Speir, James Brault Source: Science, New Series, Vol. 142, No. 3598 (Dec. 13, 1963), pp. 1485-1487 Published by: American Association for the Advancement of Science

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Shuttle-Streaming: Synchronization with Heat Production in Slime Mold

a

mold

plasmodium were allowed to fuse into

a dumbbell-shaped mass in a thermally insulated Kamiya double chamber equipped with naked bead thermistors in contact with the blobs. Cyclic tem-

perature

to

by a sensitive lock-in amplifier method with a basal noise level of less than

2

stant of 0.5 second. The temperature differences were caused by periodic

bursts of heat production synchronized perfectly with the shuttle-streaming

the

of

the streaming cytoplasm. The results support the theory that the motive force

for cytoplasmic streaming in the slime mold is pressure, probably generated by contraction of elements in the chan- nel walls.

source

cycle

Abstract.

channel

Two

small

from

blobs

a

slime

and

excised

differences

of

from

1

X

10-4

5

X

X 10O- deg Celsius were recorded

10-5 deg Celsius

and a time

con-

and

invariably

than

localized

the

at

rather

destination

The two central problems in the

cyto-

plasmic streaming phenomena at the cellular or organismal level are (i) identification of the motive force (that

analysis

of

the

mechanism

of

is, as pressure, tension, or other force), and (ii) localization of the site of its

the

case

um.

application

within

slime

the

mold,

cell,

or

in

of

the

the plasmodi-

The

solutions

to

these

problems

be-

lieved. Accounts of shuttle-streaming,

ameboid movement, cytokinesis, mi- tosis and other dynamic cellular events

"con-

human

only of observing de-

formations,

strated to have receptors for determin- ing which deformations are active or passive (1). By way of contrast, mus- cular contractions can be measured isometrically as tension, or isotonically as work performed. The present experiments, designed to localize the site of application of the motive force for shuttle-streaming in the slime mold, are based on the well- known axiom of classical muscle physi- ology, that during contraction sig- nificant amounts of heat are invariably released (2), and on the assumption that the same principle should hold true for the mechanochemical processes

are

often

not

so

simple

the

as

some

have

of

include

We

capable

description

that

the

tractions."

eye

is

claim

and has not been demon-

13 DECEMBER

1963

responsible

in the slime mold.

for

cytoplasmic

streaming

Plasmodia

of

Physarum

polycepha-

paper saturated with tap water and sprinkled

with oatmeal (3), and then allowed to

fast for a day before two blobs (about

5

diam-

eter)

into

modified

lu-

cite

small bead thermistors

diameter) would lie in contact with

the

and

two

compartments. The double chamber was then lowered into a thermally in-

sulated (styrofoam) box fitted with double-layer plastic windows adequate for observing the direction of streaming

with

were

passed through the insulated box to op- posite sides of a Wheatstone bridge

re-

was a sensitive

potentiometer

the bridge

circuit

sistances,

that

mm in

lum

were

in

cultured

diameter)

long,

on

filter

mm

and

0.2

mm

a

channel

in

(about

(4).

6 mm

were excised and allowed to fuse

a

The

dumbbell-shaped

mass

in

a

Kamiya

chambers

double

chamber

were

made

of

(Fig.

1)

and

so

designed

(5)

(0.1

terminal

blobs.

was embedded

glass

placed

The

connecting

in vaseline

over

the

channel

a cover

a

dissecting

from

containing

microscope.

the

Leads

thermistors

two

additional

one of which

for balancing

(Fig.

2).

An

oscillator

in

the

lock-in

amplifier (6) provided a 400 cy/sec modulation for the bridge. Any thermal imbalance between the two thermistors resulted in a signal at the single-ended output across the bridge. This signal was amplified by a vacuum-tube volt- meter (7) before reaching the tuned amplifier of the lock-in. The use of

a lock-in amplifier allowed two major

improvements

thermistor thermometer described by

similar

over a somewhat

Stow (8) in that the phase-sensitive detector allowed the sign of the temper- ature differences to be registered, and the narrow-band detection afforded by

a significant

this technique

reduction

The deflection sensitivity was deter-

mined by direct calibration in solutions

of

at

very

was

of

apart with

resulted

in

in the basal noise level

temperatures

a few

the

instrument

gains.

The

by

basal

(9).

known

low

degrees

level

wads

operated

noise

determined

allowing

moist

thermistors until thermal

had

gain, the rms noise was equivalent

less

the filtering time constant

in

the

cotton

been

than

to lie in contact

established.

2

X

10-5 deg

At

with the

equilibrium

maximum

to

when

Celsius

of the lock-

with that of second).

amplifier was matched

thermistors

(

-

0.5

p

Fig.

Kamiya double

sition of the thermistors

lets

thermistor leads (L).

1.

A

diagram of the modified

chamber showing the po-

(T), pressure out-

to the

(P) and terminals connected

the

present study were comparable in sensi-

tivity to the elegant thermopile-galvano-

meter measurements

in muscle and nerve by A. V. Hill and co-workers (10). The present tech-

nique was chosen

simplicity,

In

commercially in sizes considerably

smaller

interval of ther-

mal isolation was required to ensure

the onset of vigorous

streaming and to allow thermal pertur-

bations in the double side. To visualize

chromatic

to

lie outside the normal absorption range of the plasmodial pigments (220 to

440

line

of

filter, and a vari-

able-density polarizing filter adjusted to transmit barely enough light to render the direction of streaming visible.

water, an interference

Electric

AH-4

green

source

The

measurements

reported

in

of heat production

principally for its

and

are

economy.

available

blobs.

convenience

thermistors

the

addition,

than

plasmodial

A 30- to 50-minute

cytoplasmic

chamber to sub-

a mono-

chosen

streaming,

The

of

a

was

light

(11).

mt)

was

mt)

(546

mercury

General

by

2

lamp

filtered

cm

The tendency of plasmodia to mi- grate caused some to lose contact with

one

or both thermistors,

and therefore

I-

i

I

l

tUNF IVNED D

AUPL.I.lR

t

.

LOCK-IN

PozsE- t

D

i(plEsu

f

iTP-. lle

AMPLIFIER

Pll

1Crs

.

SHl*tn

T osl

;1,1ATI

I

I

Fig. 2. A block diagram of the differential thermometer.

1485

only about half of the preparations showed thermal cycles. In the remain-

ing 20 successful experiments, the tem- perature differences recorded ranged

from

Celsius. The wide differences in magni-

tude probably reflected

movement

tact with the thermistors, but a tend-

ency of some plasmodia to secrete an insulating layer of slime. Changes in the sign of the tempera- ture difference were perfectly syn-

chronized with changes in the direc-

tion of streaming. Invariably,

greater heat production was detected

at the source of the streaming

plasm (Fig. 3). It was established that

the thermal cycles were produced by the plasmodia and not by instrumental

factors

some

in the dark, there were thermal

before the illumination was turned on;

the light neither caused nor detectibly affected the thermal cycles. (ii) In con-

trol

cotton, there were no thermal cycles.

(iii) The possibility that the thermal cycles were produced by the transport away from the thermistors of cyto- plasm heated by the thermistors was eliminated by measuring the thermal

at

a constant

cycles

1

X

10-4 and

of

5

X

not

out

10-'

only

of

deg.

the

con-

plasmodia

the

cyto-

(i)

In

by

the

following

which

facts.

experiments

were initiated

wads

cycles

of

moist

experiments

with

in

by

the

same

plasmodium

sensitivity

main-

tained

tions of input voltage to the bridge and amplifier gain. No correspondence was found between the input voltage to

the

the

thermal

dium.

meas-

ured

cotton

showed

coupled

with

warmer

deflection

various

and

the

different

combina-

of

plasmo-

bridge

A

amplitude

in

the

cycles

found

of

wad

single blob

against

a

nonrhythmic

(Fig.

not

4).

plasmodium

of

moist

a gradual tendency

relative to

the

to become

cotton

of

heat

of

pro-

plas-

a

strand

a strand

bursts

Two

duction

modium

showed similar but smaller bursts of thermal activity without rhythmicity

blobs

by

connected

(Fig.

5).

Similarly,

dumbbell-shaped

thermal

plasmodia

cycles lost their rhythmicity when the connecting channel was excised. One in which

was an experiment

in

a passage the vaseline seal between the two blobs.

The observation of thermal cycles in this experiment led us to connect two plasmodial blobs with a wet cotton string, a procedure found by Kamiya and Abe (12) to permit the continuance of surface potential difference waves. Under these conditions, thermal cycles

there

exception

which

had

shown

of

remained

water

1486

of

were

not

dumbbell-shaped pl [asmodia (compare

Figs. 3 and 6). Opinions have difl fered as to whether

in

were

found (Fig. 6) all though they

in

the

so

characteristi

ic

regular

as

frequency

phenomenologically distinct from other examples of cytoplasmic streaming

and

one

un-

manifestation

cytoplasmic rotation),

(such

those

the

to

observed

slime

be

as

ameboid

of

movement,

or

to

be

(but

a universal

known) mechanism of cytoplasmic streaming. Seifriz, on observing time-

shuttle-streaming

should

mold

be

considere

:d

a process

lapse motion pictures of the "pulsa-

tions" in plasmodia (13), advanced the theory that streaming in slime molds

L was induced by pressure differences resulting from localized contractions. LLater, he abandoned this theory on the

basis that it was incompatible with the

details of streaming phenomena in

other organisms (14). Hilton (15) was

probably the first to show that the

direction of streaming could be shifted

by mechanical pressure applied to the plasmodium from outside, and he concluded that streaming was caused by "expansions and contractions" of the

plasmodial walls. More recently,

ya

truncated velocity profiles across a

main

the

that the

tem-

400.

350 0

300

250

200

150

100-

5

0

0

-

L

L I

I )x?'~ i

!

v I

2

t

L

l

I v

I

i

L

A /v

I [/ V

R

.MINUTESTE

a

ba

between

record of

reco

(x

AT

1o 5e)

Kami-

and Kuroda

channel

streaming

(16)

showed

Fig. 3. A sample of

perature

differences

thermistors the rstors

the

in contact with the t wo plasmodial blobs

(see

Fig.

1).

were

the

same,

whether

was

natural

or

induced

( x1

5

)

50

0

R,M.S. NOISE LE

EVEL2.3

2

4

6

X 10'5

by gas pressure; on this basis it was reasoned that pressure was the normal

motive force, although Kamiya (17, 18) later admitted that (i) "suction"

^ or another force acting from the front,

.

Fig.

4.

A

ferences

record of

between

a

blob and a piece of

roughly

equal

size.

MINUTES

the temperature dif-

plasmodial

of

moistened cotton

single

AT

(Xo

's5)

250

200

150

100

50

-

R.MS,.NOISE LEV

EL: 2.3 X 1-'

2

4

6

.------

a

10

12

14

16

or (ii) active shearing along thinner channel walls, could not be eliminated as an explanation. The latter (active shear- ing) was said possibly to offer a com- mon mechanism for shuttle-streaming and rotational streaming. The truncated velocity profiles would also be com-

patible with a frontal contraction mech- anism, such as that recently proposed for the ameba by Allen (19), parti-

cularly in view of some of the com-

plexities of movement that have been

pointed out by Kamiya (17) and by the

is con-

ceivable that contractions at the front

of cytoplasmic streams

cally insulated from might exert tension on structural ele-

o plasmodial

the temperature dif-

Stewarts

(20,

21).

That

is,

it

MINUTES

Fig.

5.

A

record of

ferences between tw thermally and electri

one another.

( x 10to5)

450

400

350

300

200

150

t00

50

R.M.S. N

4

6

Fig.

ferences

connected

6. A record of

between

only

by

blobis

(destination)

ments which have now been demon-

strated to exist in the axial region of the endoplasmic stream of the slime

mold

appear to estab-

(16,

21).

The

present results

4OSELEVEL 2.3 X 105

lish that if

a

single

streaming can be attributed

motive

must

force

mechanism,

the

force

force

be

much

is

in

less

to

that force must be applied where the

greater part of the heat synchronized

with the streaming cycle is produced;

stream. If

origin of

op-

eration, they must either operate in the

same general region dium, or the second

im-

not applied

of the plasmo-

more

motive

that is,

\/\

14

at the

than one

source

8

I0

12

te

--

16

-

18

MINUTES

t

the temperature dif-

blobs

cotton thread.

e

d-

tw vo plasmodial

A

a

wet

11-1_

_

_-

at the

SCIENCE,

VOL.

142

portant

portant

motive

motive

quantitatively

quantitatively

than

than

force

force

responsible

responsible

for

for

the

the

the

the

main

main

ther-

ther-

mal

mal cycles

cycles

found.

which we have found.

which

we have

While

While

Kamiya

Kamiya

the

the

(17),

(17),

possibility,

possibility,

that

that

the

the

admitted

admitted

by

by

motive

motive

force

force

8.

8.

9.

9.

factured

factured

by

by

the

the

Hewlett

Hewlett

Packard

Packard

Company,

Company,

Palo

Palo

Alto,

Alto,

Calif.

Calif.

R.

R.

W.

W.

Stow,

Stow,

Rev.

Rev.

Sci.

Sci.

Instr.

Instr.

29,

29,

774

774

(1958).

(1958).

A

A

similar

similar

technique

technique

has

has

been

been

described

described

for

for

measuring

measuring

small

small

phase

phase

retardations

retardations

of

of

el-

el-

liptically

liptically

polarized

polarized

light;

light;

see

see

R.

R.

D.

D.

Allen,

Allen,

J.

J.

Brault,

Brault,

R.

R.

D.

D.

Moore,

Moore,

J.

J. Cell Biol.

Biol.

Cell

223

18,

18, 223

(1963).

(1963).

22.

22.

23.

23.

Cell

Cell

Eds.

Eds.

Biology,

Biology,

R.

R.

(Academic

(Academic

R.

R.

D.

D.

Allen,

Allen,

presented

presented

at

at

S.

S.

the

the

Providence,

Providence,

R.I.,

R.I.,

D.

D.

Allen

Allen

Press,

Press,

New

New

and

and

N.

N.

York,

York,

Kamiya,

Kamiya,

in

in

press).

press).

Cox,

Cox,

J.

J.

D.

D.

Belcher,

Belcher,

paper

paper

Symposium

Symposium

1963.

1963.

on

on

Biorheology,

Biorheology,

N.

N.

6,

6,

in

in

Kamiya

1

Kamiya

1

(1954);

(1954);

the

the

U.S.

U.S.

and

and

W.

W.

also

also

by

by

N.

N.

Seifriz,

Seifriz,

Exptl.

Exptl.

Cell

Cell

Res.

Res.

unpublished

unpublished

films

films

shown

shown

Kamiya

Kamiya

during

during

1962

1962

and

and

might

might

be

be

due

due

to

to

interactions

interactions

the

the

smaller

smaller

streams

streams

and their

and their

between

between

channel

channel

walls

walls

cannot

cannot

be

be

rigorously

rigorously

excluded

excluded

on the basis of all the information

all

available,

available,

information

on the

basis

of

there

there

the

is

is

now

now

increasing

increasing

evidence

evidence

to

to

suggest

suggest

that

that

the

the

mechanochemical

mechanochemical

event

event

responsible

responsible

for

for

streaming

streaming

is

is

a

a

contraction

contraction

of the channel

(i)

(i)

channel

of the

wall mater-

of

of

mater-

wall

ial.

ial. For example:

example:

For

the occurrence

the occurrence

pulsations,

pulsations,

which

which

as

as

contractions;

contractions;

may

may

(ii)

(ii)

be

be

the

the

interpreted

interpreted

ability

ability

of

of

hanging

hanging

plasmodial

plasmodial

strands

strands

both

both

torsional

torsional

motions

motions

and

and

to

to

show

show

isotonic

isotonic

contractions

contractions

(23);

(23);

(iii)

(iii)

microscopi-

microscopi-

cally

cally

observable

observable

diameter

diameter

during

during

changes

changes

in

in

the

the

streaming

streaming

channel

channel

cycle;

cycle;

10.

10.

11.

11.

12.

12.

13.

13.

14.

14.

15.

15.

16.

16.

17.

17.

18.

18.

19.

19.

20.

20.

21.

21.

A.

A.

V.

V.

124,

124,

Hill,

Hill,

Proc.

Proc.

114

114

(1937);

(1937);

Roy.

Roy.

see

see

Soc.

Soc.

also

also

London,

London,

(2).

(2).

F.

F.

T.

T.

Wolf,

Wolf,

Phenomena

Phenomena

in

in

in

in

Photoperiodism

Photoperiodism

and

and

Plants

Plants

and

and

Animals,

Animals,

B.

Ser. B.

Ser.

Related

Related

AAAS

AAAS

Publ.

Publ.

No.

No.

55,

55,

R.

R.

B.

B.

Withrow,

321.

Withrow,

321.

Ed.

Ed.

ington,

N.

ington,

N.

D.C.,

D.C.,

1959),

1959),

Kamiya,

Kamiya,

and

and

S.

S.

Ab6.

Ab6.

p.

p.

J.

J.

Colloid

Colloid

149

149

(1950).

(1950).

W.

W.

Seifriz,

Seifriz,

Science

Science

86,

86,

2235

2235

(1937).

(1937).

- -

A.

A.

, ,

E.

E.

263

263

Nature

Nature

171,

171,

1136

1136

(1953).

(1953).

Hilton,

Hilton,

J.

J.

Quekett

Quekett

Microscop.

Microscop.

10,

10,

(1908).

(1908).

(Wash-

(Wash-

Sci.

Sci.

5,

5,

Club

Club

N.

N.

44,

44,

Kamiya

1 1

Kamiya

(1958).

(1958).

and

and

K.

K.

Kuroda,

Kuroda,

Protoplasma

Protoplasma

N.

N.

Kamiya,

Kamiya,

"Protoplasmic

"Protoplasmic

Streaming,"

Streaming,"

Proto-

Proto-

plasmatologia,

plasmatologia,

No.

No.

8

8

(1959),

(1959),

142-144,

142-144,

169-171.

169-171.

pp.

pp.

N.

N.

Kamiya,

Kamiya,

Ann.

Ann.

Rep.

Rep.

Sci.

Sci.

Works,

Works,

Fac.

Fac.

Osaka

Osaka

Univ.

Univ.

8,

8,

13

13

(1960),

(1960),

see

see

39.

39.

R.

R.

D.

D.

Allen,

Allen,

Exptl.

Exptl.

Cell

Cell

Res.

Res.

p.

p.

Suppl.

Suppl.

8,

8,

Sci.

Sci.

17

17

(1961).

(1961).

P.

P.

A.

A.

Exptl.

Stewart,

44

Stewart, and B. T. Stewart, Exptl.

and B.

(1959).

(1959).

T.

Stewart,

Motile

Motile

Cell

Cell

Res.

Res.

A.

A.

17,

17,

44

Stewart,

Stewart,

P.

P.

in Primitive

in Primitive

in

Systems in

Systems

24.

24.

25.

25.

18

18

1963.

1963.

At

At

a

a

symposium,

Wohlfarth-Bottermann

Wohlfarth-Bottermann

symposium,

recent

recent

H.

H.

Nakajima

Nakajima

and

and

K.

K.

presented

presented

reports

reports

of

of

polarized

polarized

light

light

and

and

electron

electron

microscopic

microscopic

studies,

zation

studies,

zation

respectively,

respectively,

of

of

the

the

fibrillar

fibrillar

organi-

organi-

of

of

myxomycete

myxomycete

plasmodia.

plasmodia.

These

These

re-

re-

ports

ports

will

will

in

in

Primitive

Primitive

Motile

Motile

Sys-

N.

Sys-

N.

terns

terns

in

in

Cell

Cell

appear

appear

Biology,

Biology,

R.

R.

D.

D.

Allen

Allen

and

and

Kamiya,

in

Kamiya,

in

Eds.

Eds.

(Academic

(Academic

Press,

this

Press,

this

New

New

York,

York,

press).

press).

Other

Other

papers

papers

in

in

volume

volume

will

will

summarize

summarize

the

the

state

state

of

of

knowledge

knowledge

regarding

regarding

various

various

protoplasmic

these

protoplasmic

these

streaming

streaming

systems.

systems.

Taken

Taken

together,

together,

accounts

accounts

seem

seem

to

to

ad-

ad-

mit

mit

greater

greater

diversity

diversity

of

of

mechanism

mechanism

at

at

the

the

cellular

cellular

level

level

than

than

might

might

have

have

been

been

expected

expected

a

a

few

few

years

years

ago.

ago.

Supported

from

Supported

from

the

the

by

by

National

National

research

research

Institutes

Institutes

grant

grant

of

of

RG-8691

RG-8691

Health.

Health.

We

We

thank

thank

Noburo

Noburo

Kamiya

Kamiya

and H.

and H.

Nakajima

Nakajima

for

for

their

their

helpful

helpful

suggestions,

suggestions,

and

and

William

William

Mad-

Mad-

dux

dux

for

for

his

his

participation

participation

in

in

the

the

early

early

stages

stages

of

of

this

this

work.

work.

October

October

1963

1963

8

8

(iv)

(iv)

the

the

demonstration

demonstration

of

of

a

a

protein

protein

system capable of both mechanochemi-

to

similar

similar to

those of muscular "contractile proteins";

those of muscular "contractile

proteins";

and

demonstra-

tion

tion

demonstra-

cal

cal

system capable

of

both mechanochemi-

reactions

reactions

recent

the recent

the

organized

organized

fibril-

fibril-

enzymatic

and enzymatic

and

of

of

finally

and finally

(v),

(v),

a

dynamically

a dynamically

lar

system

lar system

the

the

form

form

and

and birefringence

birefringence

of which fluctuates

of which fluctuates during the streaming

streaming

cycle

cycle

generalize

from

from

generalize

during

not

not

to

to

evidence

evidence

the

(24).

(24).

We

We

would

would

prefer

prefer

our

our

present

present

concerning

concerning

the

the

mechanism

mechanism

of

of

slime

slime

mold

mold

stream-

stream-

ing

ing

to

to

problems

problems

in the

the

in

ameboid

ameboid

movement,

movement,

mechanism

mechanism

of

of

reticulopodial