BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
October 30,1978
: NOT A PREREQUISITE
CHEMOTAXIS
993-997
FOR PLASMODIA COALESCENCE
IN PHYSARUM POLYCEPHALUM Wung-Wai *Department
of Biochemistry,
Tso*
The Chinese
Hong Kong and Radioisotope Received
August
and Man-Yin
Unit,
Wong
University
of Hong Kong,
University
of Hong‘Kong,
Shatin,
N.T.,
Hong Kong
11,1978 SUMMARY
Unlike pseudoplasmodium formation in Dictyostelium discoideum which involves the aggregation of amaebae chemotactically responding to pulses of cyclic AMP released from pacemaker cells, the plasmodia and presumably the microplasmodia of Physarum polycephalum coalesce without the presence of a chemotactic stimulus. INTRODUCTION Myxomycetes, Acrasiales,
the acellular
the cellular
slime
exhibit
many similar
perform
tactically
oriented
including
those
from heat
with
are washed
a non-nutrient
plasmodium
which
plasmodium
is
amaebae cyclic
free
contains
(2,3),
generate
AMP at intervals
signalling
molecules
an inward
chemotactic
(4,5)
of nutrient
movement.
cells
Myxomycetes
they
motile
to various
external (6-8,
AMP releasing signals
3 to 10 minutes to reach is
proposed
that
an humidified a pseudo-
of the pseudo-
designed
by sending
the peripherous
1, 9-12).
to form
pacemaker
(14,15).
stimuli
on filters
The formation event
phases
discodeum,
aggregate
biochemical
to
and Acrasiales
vegetative
and chemicals
(13).
cyclic
It
to be related
of Dictyostelium
solution,
the aggregation
out
thought
medium and dispersed
programmed
between diffuse
Both Their
amaebae
salt
are
in response
light
Some autonomously
population
(1).
movement
103-lo5
a well
molds,
features.
grown
buffered
indeed
communication.
molds
behavioural
When logarithmically Acrasiale,
slime
for cells out
in the
a pulse
These
pulsatile
cells
which
the signal
social
of
trigger
reception
0006-291X/78/0844-0993$01.00/0 993
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 84, No. 4, 1978
mechanism cell
BIOCHEMICAL
involves
bound
a presumed
through presumably
from
and cover
gated
or spirals
period
programmed
inward
which
moves in a co-ordinated
large
plasmodium
grows, Like
polycephalum
involves
microplasmodia
when allowed a growing
to get plasmodium
over
into
sporulates
in g.
but
coalesce, into
contact
can reach
our observation
coalescence
of plasmodia
that
outward
in
source
go
the
AMP signal, about
As a result
100 seconds of this
movement,
by a slime
the aggre-
sheath
to form pseudophasmodium of Physarum
(17)
the existing
smaller
(20).
in -P. polyceaphalum event
a widely (18).
This
on the nature
the coalescence
separated
The size
a chemotactic
plasmodium
depending
discodeum,
in
polycephalum,
multinucleated
of numerous
(2,19).
for
and the
substratrum.
14 cm diameter
attractants
we describe
a large
near
chemotactic
or spherulates
the assembly
last
surrounded
the
relayed
a cyclic
centers.
of amaebae
the aggregation
to be one of the known
these
is
the cells
movements
entirely
AMP receptor
receiving
the microplasmodium
coalesce
environment.
only
is
fashion
discodeum,
Myxomycete,
after
AMP mediated
to the aggregation
Dictylstelium studied
cyclic
cyclic
since
The pulsatile
RESEARCH COMMUNICATIONS
The signal
(15,16)
20 urn towards
pseudoplasmodium,
Similar
(12).
immediately
the center.
approximately
between
molecules
rings
a refractory
cleverly
association
phosphodiesterase
waves of expanding
AND BIOPHYSICAL
in p. Not
microplasmodia. plasmodia
also
of a coalesced Cyclic (21). is not
of the
coalesce
plasmodium
AMP has been In this required
or
reported
report, in the
in 2. polycephalum. MATERIALS
AND METHODS
The microplasmodia of p. polycephalum MSC were grown to logarithmic phase in liquid culture with adequate agitation similar to that described in Daniel and Balkwin (2,22). The harvested microplasmodia were washed three times in salt solution and dispersed onto sterile l&mu Millipore circular (pore size: 0.45 urn) laid on the surface of water agar. After approximately 15 minutes, the excess water was removed by absorption into the sunporting agar. Tow of these filter supported microplasmodia were lifted and attached onto a rectangular pre-moistened Millipore filter with one back to back to the other on both sides of the rectangular filter support. The whole support was then allowed to rest on a wire (see legend to the Figure) suspended in a humidity, light and temperature controlled environment such as a screw-cap bottle in a dark room. The microplasmodia on each circular filter coalesced in approximately one hour and the two coalesced plasmodia then migrated as two individuals. The migration pattern of these two plasmodia were observed at half hour intervals.
994
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCDSSION Chemotaxis, motile
positive
organisms
transduced
(23-28).
auxillary
its
effect
originates
the
including
acrasins folic
are
step
readily
--P polycephalum
dispersed one hour
pseudopodia.
migrate
a time
pattern
impose
period
there
is
any)
microplasmodia
layers do not
states sensitive
observation
to its
individuals acrasin;
in the
entire
that
function
This
as that
these
even molecules,
weight
products
coalesced
afterwards
On
expect
that
molecules
in
both
plasmodia
Our observation
(the
did
of
by sending
on each other.
7 hour
migra-
the patterns which
super-
are diffusable
and one rectangular documented
a
signallers.
filter
was recorded
filters
is
the microplasmodia
we would
small
recognized
disposed.
study,
In -NO instance
suggests
in Acrasiales
filter
support,
in inducing
coalescence.
used in this
the possibility
of both
Figure).
to
chemotaxis
molecular
migration
10 hours
(2 circular
or plasmodia
The plasmodia eliminating
This
that
(12),
Millipore
superimpose
than
commonly
communication
in this
and
related
In general,
small
for
operating,
they
longer
was shown in the
the three
is
that
are
independent
chemotaxis
on each other.
through if
If
described
and began
the idea
specific
quality
on each circular
in such a pattern
throughout tion
support
has been
been
is
chemoreceptor
strictly
of Aerasiales.
AMP and others a good
as the
to amaebae aggregation.
are species
diffusable, filter
which
in many
the chemoreception
and not
has never
in morphogenesis
cyclic
the rectangular
approximately
leads
(attractants)
acid,
(such
(23,24),
usefulness
chemotaxis
initial
though
repel
observed
that
chemotaxis)
and toxicants
In Acrasiales,
system
of the organism,
from physiological
significant
of the finding
in bacterial
on the physiology attract
has been widely
chemotactic
components
as nutrients
out
In spite
by a specialized
its
which
and negative,
that (29). the
study
noncoalescence
is
In Acrasiales,
formed
period
comes from
995
the
same culture,
due to different
only
pseudoplasmodia
(including
the
the amaebae is
not
coalescence
(1,30).
thus physiological state
is
The
from microplasmodia)
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Microplasmodia on each circular filter coalesced to give two separate small plasmodia migrating on both sides of the Millipore support. The migrating pattern of one plasmodium seven hours after set-up was shown on the left hand panel and that of both plasmodia were revealed by the opposite panel. Left hand panel: illumination from top; right hand panel: illumination from bottom.
showing
definitely
superimposably, however, not
no instance does not
support
an extracellular
explain
This
the nonsuperimposed
to the back
was observed
phosphodiesterase due only
in all is
to cyclic
coalescence
in contact
(30).
It
it
filter,
experiments
indeed
pattern
(31); of the
one of the plasmodia
of the
that
responsible
in the is apparent
-did
for
that
case
coalescence.
996
not
plasmodia around
the one on the back. If
both
is,
enzyme should
to migrate
with
observed
is
this
the extracellular
chemotaxis,
even though
chemotaxis
but
There
two separate
attempted.
have
migrate
event.
allowed
inhibiting
we should latter
is
coalesce
were
plasmodia
chemotaxis
reported
migration
if
to back
a state-dependent
AMP attractant,
inhibiting
the back
phosphodiesterase
as shown by the following: the support
whereby
presumably
the same enzyme individuals
required
for
were plasmodial
vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENTS This
research
was partially
supported
by University
of Hong Kong Research
Grant. REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Konijn, T.M., and Koevenig, J.L. (1971) Mycologia 63, 901-906. Tso, W.-W., and Mansour, T.E. (1975) J. Behav. Biol. 14, 499-504. Poff, K.L., and Skokut, M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2007-2010. Hato, M., Ueda, T., Kurihara, K., and Kobatake, Y. (1976) Cell Struct. Funct. 1, 269-278. Bonner, J.T., Clarke, W.W., Jr., Neeley, C.L., Jr., and Slifkin, M.K. (1950) J. Cell. Comp. Physiol. 36, 149-158. Coman, D.R., (1940) Arch. Pathol. 29, 220-228. Carlile, M.J., (1970) Gen. Microbial. 63, 221-226. Ueda, T., Muratsugu, M., Kurihara, K., and Kobatake, Y. (1976) Expt. Cell Res. 100, 337-344. Konijn, T.M., van de Meene, J.G.C., Bonner, J.T., and Barkley, D.S. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1152-1154. Pan, P., Hall, E.M., and Bonner, J.T. (1972) Nature New Biol. 237, 181-182. Keating, M.T., and Bonner, J.T. (1977) J. Bacterial. 130, 144-147. Bonner, J.T. (1977) Mycologia 69, 443-459. Raper, K.B. (1940) J. Elisha Mitchell Sci. Sot. 56, 241-282. Shaffer, B. (1962) Advan. Morphog. 2, 109-182. Gerisch, G. (1968) Curr. Top. Develop. Biol. 3, 157-197. Cohen, M.H., and Robertson, A. (1971) J. Theoret. Biol. 31, 119-130. Loomis, W.F. Jr. (1972) Nature New Biol. 240, 6-9. Rusch, H.P. (1969) Federation Proc. U.S.A. 28, 1761-1770. Guttes, E., and Guttes, S. (1967) Experientia 23, 713-715. Mohberg, J., and Rusch, H.P. (1969) J. Bacterial. 97, 1411-1418. Ueda, T., Terayama, K., Kurihara, K., and Kobatake, Y. (1975) J. Gen. Physiol. 65, 223-234. Daniel, J.W., and Baldwin, W.W. (1964) in Methods in Cell Physiology, Vol. 1 (Prescott, D.M. ed.) pp. 9-41, Academic Press, New York. Adler, J. (1969) Science 166, 1588-1597. Tso, W.-W., and Adler, J. (1974). J. Bacterial. 118, 560-576. Zigmond, S.H., and Hirsch, J.G. (1973) J. Exp. Med. 137, 387-410. Snyderman, R., Shin, S.H., Philips, J.K., Gewurz, H., and Mergenhagen, S.E. (1969) J. Immun. 103, 413-422. Hickman, C.J., and Ho, H.H. (1966) Ann. Rev. Phytopathol. 4, 195-200. Miller, R.L. (1966) J. Exp. Zool. 162, 22-44. Carlile, M.J. (1973) Ber. Deutsch. Bot. Ges. Bd. 86, S. 123-139. Garrod, D.R. (1974) J. Embryol. Exp. Morph. 32, 57-68. Chang, Y.Y. (1968) Science 160, 57-59.
997
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
October 30,1978
: NOT A PREREQUISITE
CHEMOTAXIS
993-997
FOR PLASMODIA COALESCENCE
IN PHYSARUM POLYCEPHALUM Wung-Wai *Department
of Biochemistry,
Tso*
The Chinese
Hong Kong and Radioisotope Received
August
and Man-Yin
Unit,
Wong
University
of Hong Kong,
University
of Hong‘Kong,
Shatin,
N.T.,
Hong Kong
11,1978 SUMMARY
Unlike pseudoplasmodium formation in Dictyostelium discoideum which involves the aggregation of amaebae chemotactically responding to pulses of cyclic AMP released from pacemaker cells, the plasmodia and presumably the microplasmodia of Physarum polycephalum coalesce without the presence of a chemotactic stimulus. INTRODUCTION Myxomycetes, Acrasiales,
the acellular
the cellular
slime
exhibit
many similar
perform
tactically
oriented
including
those
from heat
with
are washed
a non-nutrient
plasmodium
which
plasmodium
is
amaebae cyclic
free
contains
(2,3),
generate
AMP at intervals
signalling
molecules
an inward
chemotactic
(4,5)
of nutrient
movement.
cells
Myxomycetes
they
motile
to various
external (6-8,
AMP releasing signals
3 to 10 minutes to reach is
proposed
that
an humidified a pseudo-
of the pseudo-
designed
by sending
the peripherous
1, 9-12).
to form
pacemaker
(14,15).
stimuli
on filters
The formation event
phases
discodeum,
aggregate
biochemical
to
and Acrasiales
vegetative
and chemicals
(13).
cyclic
It
to be related
of Dictyostelium
solution,
the aggregation
out
thought
medium and dispersed
programmed
between diffuse
Both Their
amaebae
salt
are
in response
light
Some autonomously
population
(1).
movement
103-lo5
a well
molds,
features.
grown
buffered
indeed
communication.
molds
behavioural
When logarithmically Acrasiale,
slime
for cells out
in the
a pulse
These
pulsatile
cells
which
the signal
social
of
trigger
reception
0006-291X/78/0844-0993$01.00/0 993
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
Vol. 84, No. 4, 1978
mechanism cell
BIOCHEMICAL
involves
bound
a presumed
through presumably
from
and cover
gated
or spirals
period
programmed
inward
which
moves in a co-ordinated
large
plasmodium
grows, Like
polycephalum
involves
microplasmodia
when allowed a growing
to get plasmodium
over
into
sporulates
in g.
but
coalesce, into
contact
can reach
our observation
coalescence
of plasmodia
that
outward
in
source
go
the
AMP signal, about
As a result
100 seconds of this
movement,
by a slime
the aggre-
sheath
to form pseudophasmodium of Physarum
(17)
the existing
smaller
(20).
in -P. polyceaphalum event
a widely (18).
This
on the nature
the coalescence
separated
The size
a chemotactic
plasmodium
depending
discodeum,
in
polycephalum,
multinucleated
of numerous
(2,19).
for
and the
substratrum.
14 cm diameter
attractants
we describe
a large
near
chemotactic
or spherulates
the assembly
last
surrounded
the
relayed
a cyclic
centers.
of amaebae
the aggregation
to be one of the known
these
is
the cells
movements
entirely
AMP receptor
receiving
the microplasmodium
coalesce
environment.
only
is
fashion
discodeum,
Myxomycete,
after
AMP mediated
to the aggregation
Dictylstelium studied
cyclic
cyclic
since
The pulsatile
RESEARCH COMMUNICATIONS
The signal
(15,16)
20 urn towards
pseudoplasmodium,
Similar
(12).
immediately
the center.
approximately
between
molecules
rings
a refractory
cleverly
association
phosphodiesterase
waves of expanding
AND BIOPHYSICAL
in p. Not
microplasmodia. plasmodia
also
of a coalesced Cyclic (21). is not
of the
coalesce
plasmodium
AMP has been In this required
or
reported
report, in the
in 2. polycephalum. MATERIALS
AND METHODS
The microplasmodia of p. polycephalum MSC were grown to logarithmic phase in liquid culture with adequate agitation similar to that described in Daniel and Balkwin (2,22). The harvested microplasmodia were washed three times in salt solution and dispersed onto sterile l&mu Millipore circular (pore size: 0.45 urn) laid on the surface of water agar. After approximately 15 minutes, the excess water was removed by absorption into the sunporting agar. Tow of these filter supported microplasmodia were lifted and attached onto a rectangular pre-moistened Millipore filter with one back to back to the other on both sides of the rectangular filter support. The whole support was then allowed to rest on a wire (see legend to the Figure) suspended in a humidity, light and temperature controlled environment such as a screw-cap bottle in a dark room. The microplasmodia on each circular filter coalesced in approximately one hour and the two coalesced plasmodia then migrated as two individuals. The migration pattern of these two plasmodia were observed at half hour intervals.
994
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCDSSION Chemotaxis, motile
positive
organisms
transduced
(23-28).
auxillary
its
effect
originates
the
including
acrasins folic
are
step
readily
--P polycephalum
dispersed one hour
pseudopodia.
migrate
a time
pattern
impose
period
there
is
any)
microplasmodia
layers do not
states sensitive
observation
to its
individuals acrasin;
in the
entire
that
function
This
as that
these
even molecules,
weight
products
coalesced
afterwards
On
expect
that
molecules
in
both
plasmodia
Our observation
(the
did
of
by sending
on each other.
7 hour
migra-
the patterns which
super-
are diffusable
and one rectangular documented
a
signallers.
filter
was recorded
filters
is
the microplasmodia
we would
small
recognized
disposed.
study,
In -NO instance
suggests
in Acrasiales
filter
support,
in inducing
coalescence.
used in this
the possibility
of both
Figure).
to
chemotaxis
molecular
migration
10 hours
(2 circular
or plasmodia
The plasmodia eliminating
This
that
(12),
Millipore
superimpose
than
commonly
communication
in this
and
related
In general,
small
for
operating,
they
longer
was shown in the
the three
is
that
are
independent
chemotaxis
on each other.
through if
If
described
and began
the idea
specific
quality
on each circular
in such a pattern
throughout tion
support
has been
been
is
chemoreceptor
strictly
of Aerasiales.
AMP and others a good
as the
to amaebae aggregation.
are species
diffusable, filter
which
in many
the chemoreception
and not
has never
in morphogenesis
cyclic
the rectangular
approximately
leads
(attractants)
acid,
(such
(23,24),
usefulness
chemotaxis
initial
though
repel
observed
that
chemotaxis)
and toxicants
In Acrasiales,
system
of the organism,
from physiological
significant
of the finding
in bacterial
on the physiology attract
has been widely
chemotactic
components
as nutrients
out
In spite
by a specialized
its
which
and negative,
that (29). the
study
noncoalescence
is
In Acrasiales,
formed
period
comes from
995
the
same culture,
due to different
only
pseudoplasmodia
(including
the
the amaebae is
not
coalescence
(1,30).
thus physiological state
is
The
from microplasmodia)
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Microplasmodia on each circular filter coalesced to give two separate small plasmodia migrating on both sides of the Millipore support. The migrating pattern of one plasmodium seven hours after set-up was shown on the left hand panel and that of both plasmodia were revealed by the opposite panel. Left hand panel: illumination from top; right hand panel: illumination from bottom.
showing
definitely
superimposably, however, not
no instance does not
support
an extracellular
explain
This
the nonsuperimposed
to the back
was observed
phosphodiesterase due only
in all is
to cyclic
coalescence
in contact
(30).
It
it
filter,
experiments
indeed
pattern
(31); of the
one of the plasmodia
of the
that
responsible
in the is apparent
-did
for
that
case
coalescence.
996
not
plasmodia around
the one on the back. If
both
is,
enzyme should
to migrate
with
observed
is
this
the extracellular
chemotaxis,
even though
chemotaxis
but
There
two separate
attempted.
have
migrate
event.
allowed
inhibiting
we should latter
is
coalesce
were
plasmodia
chemotaxis
reported
migration
if
to back
a state-dependent
AMP attractant,
inhibiting
the back
phosphodiesterase
as shown by the following: the support
whereby
presumably
the same enzyme individuals
required
for
were plasmodial
vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENTS This
research
was partially
supported
by University
of Hong Kong Research
Grant. REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
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