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STUDIES ON M I C R O P L A S M O D I A OF P H Y S A R U M P O L Y C E P H A L U M V: E L E C T R I C A L A C T I V I T Y OF D I F F E R E N T TYPES OF MICROP L A S M O D I A AND M A C R O P L A S M O D I A
R. Meyer and W. S t o c k e m Institute of Cytology, U n i v e r s i t y of Bonn D - 5 3 O Q Bonn I, U l r i c h - H a b e r l a n d - S t r . 61 a Federal Republic of Germany
ABSTRACT Results o b t a i n e d by three d i f f e r e n t e l e c t r o p h y s i o l o g i c a l m e t h o d s d e l i v e r e d m a i n l y o s c i l l a t i n g p o t e n t i a l s (indep e n d e n t l y of the type of o r g a n i z a t i o n of P h y s a r u m polycephalum) with an average period length of I min 21 sec. This value is in good statistical agreement with corr e s p o n d i n g data for the p e r i o d i c a l o s c i l l a t i o n of the shuttle streaming (I min 47 sec) and coincides also with the radial t e n s i o m e t r i c c o n t r a c t i l e activity (I min 34 sec). The i n v e s t i g a t i o n favours the h y p o t h e s i s that ion fluxes across the cell m e m b r a n e and perhaps b e t w e e n i n t r a c e l l u l a r c o m p a r t m e n t s may act as a trigger mechanism for a still u n k n o w n o s c i l l a t o r - s y s t e m which controls cell m o v e m e n t p h e n o m e n a in the acellular slime molds.
INTRODUCTION The m o t i v e force for the g e n e r a t i o n of the highly organized p r o t o p l a s m i c streaming p a t t e r n of the a c e l l u l a r slime m o l d P h y s a r u m p o l y c e p h a l u m is produced by the activity of a c o n t r a c t i l e a c t o m y o s i n system (Kamiya, 1959; W o h l f a r t h - B o t t e r m & n n , 1975a). A l t h o u g h recent progress has been made in the m o l e c u l a r (Hatano and Oosawa, 1966) and in the fine structural i d e n t i f i c a t i o n (WohlfarthBottermann, 1962, 1963) and e x p l a n a t i o n (Wohlfarth-Bottermann, 1975 b and c) of this system, the control mechanism for the p h e n o m e n o n of shuttle streaming, i.e. the c o o r d i n a t e d c o n t r a c t i o n and r e l a x a t i o n of the a c t o m y o s i n apparatus in d i f f e r e n t regions of the p l a s m o d i a l network, remains still to be clarified. F u n c t i o n a l and morp h o l o g i c a l similarities b e t w e e n the c y t o p l a s m i c and the m u s c u l a r actomyosins (D'Haese and Hinssen, 1978) were supposed to be a suitable indication for the fact that 0309--1651/79/040321 --10/$02.00/0
(~)1979 Academic Press Inc. (London) Ltd.
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ion fluxes across the cell membrane could trigger the activity of the contractile proteins in the acellular slime molds. Kamiya and Ab~ (1950) first succeeded in measuring rhythmic potentials in Physarum p o l y c e p h a l u m by the double chamber method, which was also used by following investigators (Ueda and Kobatake, 1978). Rhea (1966) obtained similar data by intracellular measurements with conventional microelectrodes. These results d e m o n s t r a t e d the feasible correlation between electrical activities of the plasma membrane on the one hand and the c o n t r a c t i o n - r e l a x a t i o n cycle of the actomyosin system on the other. Physiological experiments with tensiometric techniques,A however, failed to prove the existence of controlled C a 2 ~ f l u x e s between the environment and the cell interior (Wohlfarth-Bottermann and G~tz von Olenhusen, 1977). Therefore, we r e i n v e s t i g a t e d the question for the participation of the cell membrane in triggering protoplasmic streaming activity in m i c r o p l a s m o d i a and m a c r o p l a s m o d i a of Physarum polycephalum. Three different procedures were used in order to exclude methodical mistakes. MATERIAL
AND METHODS
M i c r o p l a s m o d i a and m a c r o p l a s m o d i a of the acellular slime mold Physarum p o l y c e p h a l u m were grown in axenic cultures (Daniel and Rusch, 1961), on agar, and on filter paper (Camp, 1936). The following methods were used for the registration of electrical activities: I. Changes of the cell surface potential were m e a s u r e d with a platin electrode (tip diameter 20 ~m) set on single veins. The reference electrode was inserted into the substrate (agar) and connected to ground. Simultaneously the activity of the contractile apparatus was recorded tensiometrically. Control experiments were done with the same equipment on veins isolated from the tip of the platin electrode by a thin plastic foil. 2. Ion fluxes across the plasma membrane were demonstrated by a method recently propagated by Chen (1977) on S p i r o s t o m u m ambiguum. Two different types of fluorinert liquid (FC 43 and FC 70, 3-M Company) were used for the electrical isolation of single veins (diameter 0.5 mm, length 3 mm). The fluorinert liquids are clear, colourless and not toxic. They can be saturated with oxygen (0.282 p/n/ml), are of low v i s c o s i t y (2 ~ centi ~tockes) and posess a specific resistance of i01 O h m / c m . Single veins of Physarum p o l y c e p h a l u m survive in fluorinert liquid for several hours without essential changes in shuttle streaming and contractile activity. The procedure of preparing and measuring veins is shown in fig. 1. The veins were isolated as usual (figla,b) and air dried
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for 30 - 60 seconds. Subsequently they were put into a chamber containing fluorinert liquid (fig; id). Two small droplets of distilled water were applied to both ends of the vein. M i c r o e l e c t r o d e s filled with 3 M KCI and inserted into the water droplets were used to record changes in ion content as described in fig. ic. The shuttle streaming was recorded simultaneously by light m i c r o s c o p i c a l observation. a b
C
4
5
6
7
8
4
13
d
j 8
/..
.
.
.
.
5
6
7
8
12
I
10
Fig. I: Preparation and method of m e a s u r e m e n t in fluorinert liquids. I. Substrate (agar), 2. plasmodium, 3. needle, 4. separated vein, 5. water droplet, 6. electrode, 7. bath (fluorinert liquid), 8. o b s e r v a t i o n chamber, 9. entrance amplifier, iO. marker to record the shuttle streaming, ii. pen recorder, 12. compensation, 13. difference amplifier. a) A m p u t a t i o n of the vein b) A i r - d r y i n g of the vein c) Hypothetic ion-fluctuations during the m e a s u r e m e n t d) Total setup for the experiments
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3, The membrane potential of m i c r o p l a s m o d i a and macroplasmodia was m e a s u r e d intracellularly with conventional glass microelectrodes. The 3 M KCI solution contained I% EDTA (Rhea, 1966; Hato et al., 1976), 1% Triton, or 1% acetone as detergents. These detergents should prevent cell membrane regeneration of Physarum, which is supposed to occur during intracellular m e a s u r e m e n t s (Kamiya and Abe, 1950; Kishimoto, 1958; Rhea, 1966; Olfers-Weber et al., 1976). The addition of 1% EDTA gave the best results. The tip resistance of m i c r o e l e c t r o d e s prepared in this way ranged from i0 to iOO M Ohm; the outside diameter varied between O.i and 0.2 ~m. It was controlled by scanning electron microscopy. The veins were m e a s u r e d in situ on agar, whereas the m i c r o p l a s m o d i a were covered by a stabilizing solution (Hato et al., 1976) before the m i c r o e l e c t r o d e was inserted into the protoplasm. In all cases, the shuttle streaming a c t i v i t y was recorded sim u l t a n e o u s l y by light microscopy. RESULTS
AND DISCUSSION
I. Measurements of the cell surface potential M a c r o p l a s m o d i a m e a s u r e d by this method (n=15) showed rhythmic alterations of the cell surface potential (fig. 2, PD) with minimum and m a x i m u m amplitudes between I and 6 mY. The mean value of the potential level was found to be characteristic for each m a c r o p l a s m o d i u m and ranged from 8 to 20 mY. It remained constant over very long periods (up to iOO min). The average value of the periodicity amounted to I min 46 sec (~ SD 20 sec). In some cases the period of the surface potential was correlated with the tensiometric radial activity m e a s u r e d simultaneously (see fig. 2 F).
F imp] PD Im8~[I
F
-/,0'
F
PD
1
10
20
30
t.0
/.6 t [rain]
Fig. 2: Simultaneous recording of the isometrically measured radial contractile activity (F) and of the surface potential (PD). The period length of both waves is nearly the same but there is a phase difference of half of a period (scale: 0 to 80=F IMP]; 0 to -40=PD [mV]).
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F r o m control experiments it could be excluded that the m e a s u r e d o s c i l l a t i o n s were produced a r t i f i c i a l l y by mechanical forces (e.g. radial c o n t r a c t i o n s of the veins, W o h l f a r t h - B o t t e r m a n n , 1975b) acting on the tip of the electrode. The electrical isolation of the veins (see m a t e r i a l and methods) caused the c e s s a t i o n of the surface potential but not of the t e n s i o m e t r i c activity. 2. M e a s u r e m e n t s in f l u o r i n e r t liquids Isolated veins i n v e s t i g a t e d in fluorinert liquids (n=lO) showed very r e g u l a r potential o s c i l l a t i o n s (fig. 3a,b). Both liquids (FC 43 and FC 70) proved to be a p p r o p r i a t e and allowed r e c o r d i n g s between 20 and 35 min. Later on, the water droplets fused and thereby p r o d u c e d a short circuit. The average period length of the shuttle streaming and of the s i m u l t a n e o u s l y m e a s u r e d p o t e n t i a l was compared by Student's T-test. Both the electrical potential (x=! min 15 sec, ±SD 18 sec) and the shuttle streaming (x=1 min 14 sec, ~SD 19 sec) were of the same order of m a g n i t u d e w i t h o u t any s i g n i f i c a n t difference. In a second T-test the question of a temporal correlation b e t w e e n the points of the m i n i m u m and m a x i m u m potential values on the one hand and the change of protoplasmic streaming d i r e c t i o n on the other hand was answered. In 93% of all cases analysed such a c o r r e l a t i o n existed. The potential d i f f e r e n c e s o b t a i n e d by this m e t h o d can be caused either by rhythmic ion fluctuations b e t w e e n the slime mold and the d i s t i l l e d water or by the action of a b i o e l e c t r i c field p r o d u c e d in the mold. C o n t r o l l e d rhythmic ion fluxes across the plasma m e m b r a n e seem to be the most p r o b a b l e and i l l u m i n a t i n g e x p l a n a t i o n for the results d e l i v e r e d by the fluorinert liquid technique (c.f. the results of Ueda and Kobatake, 1977 as well as Rhea, 1966, obtained with other methods). [mY]
Fig.
3
3a
.
÷2"
i
.
.
.
.
.
.
.
.
.
.
.
.
.
.
|i
2o t[min]
PD [mY]
3b 0 .2
~~
t [rain]
Fig. 3: Two recordings of s e p a r a t e d veins in fluorinert liquid. The shuttle streaming is noted as square wave. Both r e c o r d i n g s show c o r r e l a t i o n b e t w e e n streaming and potential oscillations.
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3. M e a s u r e m e n t s w i t h intracellular m i c r o e l e c t r o d e s The addition of E D T A to the K C l - c o n t e n t of the m i c r o e l e trode allowed intracellular m e a s u r e m e n t s (between 5 and 72 min) in m a c r o p l a s m o d i a (n=ll) and d i f f e r e n t types of m i c r o p l a s m o d i a (n=35), (Gawlitta and Stockem, in preparation). Half of the obtained recordings d e m o n s t r a t e d the existence of more or less rhythmic potential oscillations (fig. 4 a,b,c,d,e) in both cells with (fig. 4 b, c,d,e) and w i t h o u t (fig. 4 a) any p r o t o p l a s m i c streaming activity. On the other hand irregular or c o m p l e t e l y smooth curves could be r e c e i v e d from macro- or microplasmodia, showing normal p r o t o p l a s m i c shuttle streaming (fig. 4 f). A r e l a t i o n s h i p b e t w e e n the extreme values of rhythmic potential alterations and the points of change of p r o t o p l a s m i c streaming d i r e c t i o n existed to some extent (fig. 4 c,d,e). The average value for the level of the m e m b r a n e potential amounted to 53.6 mY ±SD 9.9 mV and the m a i n period length in periodic recordings was c a l c u l a t e d as I min +SD 21 sec. The obtained aperiodic r e c o r d i n g s are very d i f f i c u l t to explain. Besides m e t h o d i c a l reasons (leak current along the surface of the electrode), other cell p h y s i o l o g i c a l processes (intracellular ion fluxes between d i f f e r e n t subcellular compartments) could be superimposed on the regular i o n - e x c h a n g e across the m e m b r a n e and thus c o n t r i b u t e to the i r r e g u l a r i t y of the curves. CONCLUDING RE~RKS The existence of o s c i l l a t i n g electrical activities in micro- and m a c r o p l a s m o d i a of P h y s a r u m p o l y c e p h a l u m could be d e m o n s t r a t e d with each of the three m e t h o d s tested in this investigation. This confirms the a s s u m p t i o n that rhythmic ion fluxes across the cell m e m b r a n e may play a very important role in the life cycle of this o r g a n i s m (Durham, 1974). The similar period length of the oscillating potential activity m e a s u r e d in each series of experiments (fig. 5), as well as the good a g r e e m e n t of the average value of this period length (1.21 min) with both the values for the d u r a t i o n of p r o t o p l a s m i c streaming (1.47 min, H ~ i s m a n n and W o h l f a r t h - B o t t e r m a n n , 1978) a n d radial t e n s i o m e t r i c activity (1.34 min, W o h l f a r t h Bottermann, 1977), can also be c o n s i d e r e d to support this suggestion. The fact that the o s c i l l a t i n g potential alterations could be m e a s u r e d in all types of m i c r o p l a s m o d i a - ind e p e n d e n t l y o f the existence of p r o t o p l a s m i c shuttle streaming - excludes the g e n e r a t i o n of p e r i o d i c a l ion fluxes across the cell m e m b r a n e as a c o n s e q u e n c e of the streaming activity. Regular ion exchanges could rather indicate the existence of a s u p e r i m p o s e d cytoplasmic regulatory system t r i g g e r i n g the rhythmic c o n t r a c t i o n -
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PD [_m~l
-10 1
10
20
30
40
44
't [min}
PD [mV] -70
b -10 :
:
:
:
:
:
:
:
:
:
i
i
I
i
i
I0
i
i
i
20
i
i
i
i
i
i
i
i
,
j
. . . . . . . .
.
30
.
40
.
.1
43 t [rain}
PD [mV] -90PD
[mV] - 70
C
•
e 1
-lO
i 10
r
PD [mV] -80
16
PD [mY -90
~
-lo t [mini
1
rUU~
10
r
19
t [min]
~
d -10
-10 '
I
I
I
~
'
' ~ g
t lmin]
: : : ; i 1 1
. . . . . . . . . . . . 10
20
oi 27
t [mini
Fig. 4. Intracellular m e m b r a n e potential recordings from different types of plasmodia. a) Rhythmic potential of a sphaerical microplasmodium. b) Rhythmic potential of an amoeboid microplasmodium. c) Rhythmic potential of a fused microplasmodium. d) Rhythmic potential of a single vein of a macroplasmodium. e) Rhythmic potential of a d u m p b e l l - s ~ a p e d microplasmodium. f) Aperiodic potential of a d u m p b e l l - s h a p e d m i c r o p l a s m o dium.
Cell Biology International Reports, Vol. 3, No. 4, 1979
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relaxation cycle of the actomyosin system (Ridgway and Durham, 1976; Kato, 1977; Ueda et al., 1978). It seems to be possible and should be proved by further experiments that ion fluxes across the cell membrane can modulate the activity of this regulatory system (e.g. by changing the period of the oscillation). Thereby they could transfer external stimuli to the cell interior, which is a necessary precondition for chemotactical reactions in general. Fig. 5: Statistical variations of the period length of electrical activity in different plasmodia
/
~oo 11o m 1
1. Measurements in fluorinert 2. M e a s u r e m e n t s
of the cell
3. Measurements
with
m>l~o sec
liquids surface
intracellular
potential microelectrodes
ACKNOWLEDGMENT We thank Mr. E. Samans for technical assistance and Prof. Dr. K.E. W o h l f a r t h - B o t t e r m a n n for providing the tensiometer equipment as well as for discussion.
REFERENCES Camp,
W.G. (1936) A method of cultivating myxomycete plasmodia. Bulletin of the Torrey Botanical Club, 63, 205. Chen, V.K.-H. (1977) Recording of m e m b r a n e potential change in Spirostomum ambiguum correlated with mechanically stimulated rapid body contraction. Abstracts of P a p e ~ r e a d at the Fifth International Congress on Protozoology, S.H. Hutner ed., 305. Daniel, J.W. and Rusch, H.F. (1961) The pure culture of Physarum p o l y c e p h a l u m on a partially soluble medium. Journal of the General Microbiology, 25, 47-59.
Cell Biology International Reports, Vol. 3, No. 4, 1979
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D'Haese, J. and Hinssen, H. (1978) Kontraktionseigenschaften von isoliertem Schleimpilzactomyosin. I. Vergleichende Untersuchungen von Fadenmodellen aus natHrlichen, rekombinierten und hybridisierten Actomyosinen von Schleimpilz und Muskel. Protoplasma, 95, 273-295. Durham, A.C.H. (1974) A unified theory of the control of actin and myosin in non muscle movements. Cell, 2, 123-136. Hatano, S. and Oosawa, F. (1966) Isolation and characterization of plasmodium actin. Biochimica et Biophysica Acta, 127, 488-498. Hato, M., Ueda, T., Kurihara, K., and Kobatake, Y. (1976) Change in zeta potential and membrane potential of slime mold Physarum polycephalum in response to chemical stimuli. Biochimica et Biophysica Acta, 426, 73-80. HHlsmann, N. and Wohlfarth-Bottermann, K,E. (1978) Spatio-temporal relationship between protoplasmic streaming and contraction activities in plasmodial veins of Physarum polycephalum. Cytobiologie, 17, 317-334, 1978. Kamiya, N. and Abe, S. (1950) Bioelectronic phenomena in the myxomycete plasmodium and their relation to protoplasmic flow. Journal of Colloid and Interface Sciences, 5, 149-163. Kamiya, N. (1959) Protoplasmic streaming. Protoplasmatologia VIII/3/a, ~Heilbrunn, L.V., F. Weber ed., Wien, Springer Verlag Berlin, Heidelberg, New York, 1-199. Kato, T. and Tonomura, Y. (1977) Uptake of calcium ions into microsomas isolated from Physarum polycephalum. Journal of Biochemistry, 81, 207-213. Kishimoto, U. (1958) Rhythmicity in the protoplasmic streaming of a slime mold Physarum polycephalum. I. A statistical analysis of the electric potential rhythm. II. Theoretical treatment of the electric potential rhythm. Journal of General Physiology, 41, 1205-1245. Olfers-Weber, R., Stockem, W. and Wohlfarth-Bottermann, E.(1976) Cytological aspects of membrane regeneration after experimental injury of amebae and acellular slime molds. Ion and enzyme electrodes in biology and medicine. M. Kessler, L.C.Clark, D.W. LHbbers, i.A. Silver, W. Simon eds., Urban & S c h w a r z e n b e r g MHnchen, Berlin, Wien, 205-216. Rhea, R.P. (1966) Microcinematographic, electron microscopic and electrophysiological studies on shuttle streaming in the slime mold Physarum polycephalum. Dynamic of fluids and plasmas. S.I. Pai et al. eds, Academic Press Inc. New York, 35-58.
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Ridgway, E.B. and Durham, A.C.H. (1976) Oscillations of calcium ion concentrations in Physarum polycephalum. Journal of Cell Biology, 69, 223-226. Ueda, T. and Kobatake, Y. (1977) Changes in membrane potential, zeta potential and chemotaxis of Physarum polycephalum in response to n-alcohols, naldehydes and n-fatty acids. Cytobiologie, 16, 16-26. Ueda, T., G~tz von Olenhusen, K., Wohlfarth-Bottermann, K.E. (1978) Reaction of the ~ n t r a c t i l e apparatus in Physarum to injected Ca--, ATP, ADP and 5'AMP. Cytobiologie, 18, 76-94. Wohlfarth-Bottermann, K.E. (1962) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung fHr die Protoplasmastr~mung. I. Elektronenmikroskopischer Nachweis und Feinstruktur. Protoplasma, 54, 514-539. Wohlfarth-Bottermann, K.E. (1963) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung fur die Protoplasmastr~mung. II. Lichtmikro~ skopische Darstellung. Protoplasma, 57, 747-761. Wohlfarth-Bottermann, K.E. (1975a) Ursachen von Zellbewegungen, cytoplasmatische Actomyosine und ihre Bedeutung fHr Protoplasmastr6mungen und Zellmotilit~t. Vortrag 1975, Halle/Saale, Sitzung der Deutschen Akademie der Naturforscher Leopoldina. Leopoldina-Mitteilungen,21, 85-128. Wohlfarth-Bottermann, K.E. (1975b) Tensiometric demonstration of endogenous oscillating contractions in plasmodia of Physarum polycephalum. Zeitschrift fHr Pflanzenphysiologie, 76, 14-27. Wohlfarth-Bottermann, K.E. (1975c) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung f~r die Protoplasmastr~mung. X. Die Anordnung der Aktomyosin-Fibrillen in experimentell unbeeinfluBten Protoplasma-Adern von Physarum in situ. Protistologica, XI, 19-30. Wohlfarth-Bottermann, K.E. (1977) Oscillating contractions in protoplasmic strands of Physarum: Simultaneous tensiometry of longitudinal and radial rhythms, periodicity analysis and temperature dependence. Journal of Experimental Biology, 67, 49-59. Wohlfarth-Bottermann, K.E. and G~tz von Olenhusen, K. (1977) Oscillating contractions in protoplasmic strands of P h y ~ r u m : Effects of external Ca---depletion and Ca---antagonistic drugs on intrinsic contraction automaticity. Cell Biology International Reports, l, 239-247. Received:
16th November 1 9 7 9
Accepted: 22nd January 1979
321
STUDIES ON M I C R O P L A S M O D I A OF P H Y S A R U M P O L Y C E P H A L U M V: E L E C T R I C A L A C T I V I T Y OF D I F F E R E N T TYPES OF MICROP L A S M O D I A AND M A C R O P L A S M O D I A
R. Meyer and W. S t o c k e m Institute of Cytology, U n i v e r s i t y of Bonn D - 5 3 O Q Bonn I, U l r i c h - H a b e r l a n d - S t r . 61 a Federal Republic of Germany
ABSTRACT Results o b t a i n e d by three d i f f e r e n t e l e c t r o p h y s i o l o g i c a l m e t h o d s d e l i v e r e d m a i n l y o s c i l l a t i n g p o t e n t i a l s (indep e n d e n t l y of the type of o r g a n i z a t i o n of P h y s a r u m polycephalum) with an average period length of I min 21 sec. This value is in good statistical agreement with corr e s p o n d i n g data for the p e r i o d i c a l o s c i l l a t i o n of the shuttle streaming (I min 47 sec) and coincides also with the radial t e n s i o m e t r i c c o n t r a c t i l e activity (I min 34 sec). The i n v e s t i g a t i o n favours the h y p o t h e s i s that ion fluxes across the cell m e m b r a n e and perhaps b e t w e e n i n t r a c e l l u l a r c o m p a r t m e n t s may act as a trigger mechanism for a still u n k n o w n o s c i l l a t o r - s y s t e m which controls cell m o v e m e n t p h e n o m e n a in the acellular slime molds.
INTRODUCTION The m o t i v e force for the g e n e r a t i o n of the highly organized p r o t o p l a s m i c streaming p a t t e r n of the a c e l l u l a r slime m o l d P h y s a r u m p o l y c e p h a l u m is produced by the activity of a c o n t r a c t i l e a c t o m y o s i n system (Kamiya, 1959; W o h l f a r t h - B o t t e r m & n n , 1975a). A l t h o u g h recent progress has been made in the m o l e c u l a r (Hatano and Oosawa, 1966) and in the fine structural i d e n t i f i c a t i o n (WohlfarthBottermann, 1962, 1963) and e x p l a n a t i o n (Wohlfarth-Bottermann, 1975 b and c) of this system, the control mechanism for the p h e n o m e n o n of shuttle streaming, i.e. the c o o r d i n a t e d c o n t r a c t i o n and r e l a x a t i o n of the a c t o m y o s i n apparatus in d i f f e r e n t regions of the p l a s m o d i a l network, remains still to be clarified. F u n c t i o n a l and morp h o l o g i c a l similarities b e t w e e n the c y t o p l a s m i c and the m u s c u l a r actomyosins (D'Haese and Hinssen, 1978) were supposed to be a suitable indication for the fact that 0309--1651/79/040321 --10/$02.00/0
(~)1979 Academic Press Inc. (London) Ltd.
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ion fluxes across the cell membrane could trigger the activity of the contractile proteins in the acellular slime molds. Kamiya and Ab~ (1950) first succeeded in measuring rhythmic potentials in Physarum p o l y c e p h a l u m by the double chamber method, which was also used by following investigators (Ueda and Kobatake, 1978). Rhea (1966) obtained similar data by intracellular measurements with conventional microelectrodes. These results d e m o n s t r a t e d the feasible correlation between electrical activities of the plasma membrane on the one hand and the c o n t r a c t i o n - r e l a x a t i o n cycle of the actomyosin system on the other. Physiological experiments with tensiometric techniques,A however, failed to prove the existence of controlled C a 2 ~ f l u x e s between the environment and the cell interior (Wohlfarth-Bottermann and G~tz von Olenhusen, 1977). Therefore, we r e i n v e s t i g a t e d the question for the participation of the cell membrane in triggering protoplasmic streaming activity in m i c r o p l a s m o d i a and m a c r o p l a s m o d i a of Physarum polycephalum. Three different procedures were used in order to exclude methodical mistakes. MATERIAL
AND METHODS
M i c r o p l a s m o d i a and m a c r o p l a s m o d i a of the acellular slime mold Physarum p o l y c e p h a l u m were grown in axenic cultures (Daniel and Rusch, 1961), on agar, and on filter paper (Camp, 1936). The following methods were used for the registration of electrical activities: I. Changes of the cell surface potential were m e a s u r e d with a platin electrode (tip diameter 20 ~m) set on single veins. The reference electrode was inserted into the substrate (agar) and connected to ground. Simultaneously the activity of the contractile apparatus was recorded tensiometrically. Control experiments were done with the same equipment on veins isolated from the tip of the platin electrode by a thin plastic foil. 2. Ion fluxes across the plasma membrane were demonstrated by a method recently propagated by Chen (1977) on S p i r o s t o m u m ambiguum. Two different types of fluorinert liquid (FC 43 and FC 70, 3-M Company) were used for the electrical isolation of single veins (diameter 0.5 mm, length 3 mm). The fluorinert liquids are clear, colourless and not toxic. They can be saturated with oxygen (0.282 p/n/ml), are of low v i s c o s i t y (2 ~ centi ~tockes) and posess a specific resistance of i01 O h m / c m . Single veins of Physarum p o l y c e p h a l u m survive in fluorinert liquid for several hours without essential changes in shuttle streaming and contractile activity. The procedure of preparing and measuring veins is shown in fig. 1. The veins were isolated as usual (figla,b) and air dried
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for 30 - 60 seconds. Subsequently they were put into a chamber containing fluorinert liquid (fig; id). Two small droplets of distilled water were applied to both ends of the vein. M i c r o e l e c t r o d e s filled with 3 M KCI and inserted into the water droplets were used to record changes in ion content as described in fig. ic. The shuttle streaming was recorded simultaneously by light m i c r o s c o p i c a l observation. a b
C
4
5
6
7
8
4
13
d
j 8
/..
.
.
.
.
5
6
7
8
12
I
10
Fig. I: Preparation and method of m e a s u r e m e n t in fluorinert liquids. I. Substrate (agar), 2. plasmodium, 3. needle, 4. separated vein, 5. water droplet, 6. electrode, 7. bath (fluorinert liquid), 8. o b s e r v a t i o n chamber, 9. entrance amplifier, iO. marker to record the shuttle streaming, ii. pen recorder, 12. compensation, 13. difference amplifier. a) A m p u t a t i o n of the vein b) A i r - d r y i n g of the vein c) Hypothetic ion-fluctuations during the m e a s u r e m e n t d) Total setup for the experiments
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3, The membrane potential of m i c r o p l a s m o d i a and macroplasmodia was m e a s u r e d intracellularly with conventional glass microelectrodes. The 3 M KCI solution contained I% EDTA (Rhea, 1966; Hato et al., 1976), 1% Triton, or 1% acetone as detergents. These detergents should prevent cell membrane regeneration of Physarum, which is supposed to occur during intracellular m e a s u r e m e n t s (Kamiya and Abe, 1950; Kishimoto, 1958; Rhea, 1966; Olfers-Weber et al., 1976). The addition of 1% EDTA gave the best results. The tip resistance of m i c r o e l e c t r o d e s prepared in this way ranged from i0 to iOO M Ohm; the outside diameter varied between O.i and 0.2 ~m. It was controlled by scanning electron microscopy. The veins were m e a s u r e d in situ on agar, whereas the m i c r o p l a s m o d i a were covered by a stabilizing solution (Hato et al., 1976) before the m i c r o e l e c t r o d e was inserted into the protoplasm. In all cases, the shuttle streaming a c t i v i t y was recorded sim u l t a n e o u s l y by light microscopy. RESULTS
AND DISCUSSION
I. Measurements of the cell surface potential M a c r o p l a s m o d i a m e a s u r e d by this method (n=15) showed rhythmic alterations of the cell surface potential (fig. 2, PD) with minimum and m a x i m u m amplitudes between I and 6 mY. The mean value of the potential level was found to be characteristic for each m a c r o p l a s m o d i u m and ranged from 8 to 20 mY. It remained constant over very long periods (up to iOO min). The average value of the periodicity amounted to I min 46 sec (~ SD 20 sec). In some cases the period of the surface potential was correlated with the tensiometric radial activity m e a s u r e d simultaneously (see fig. 2 F).
F imp] PD Im8~[I
F
-/,0'
F
PD
1
10
20
30
t.0
/.6 t [rain]
Fig. 2: Simultaneous recording of the isometrically measured radial contractile activity (F) and of the surface potential (PD). The period length of both waves is nearly the same but there is a phase difference of half of a period (scale: 0 to 80=F IMP]; 0 to -40=PD [mV]).
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F r o m control experiments it could be excluded that the m e a s u r e d o s c i l l a t i o n s were produced a r t i f i c i a l l y by mechanical forces (e.g. radial c o n t r a c t i o n s of the veins, W o h l f a r t h - B o t t e r m a n n , 1975b) acting on the tip of the electrode. The electrical isolation of the veins (see m a t e r i a l and methods) caused the c e s s a t i o n of the surface potential but not of the t e n s i o m e t r i c activity. 2. M e a s u r e m e n t s in f l u o r i n e r t liquids Isolated veins i n v e s t i g a t e d in fluorinert liquids (n=lO) showed very r e g u l a r potential o s c i l l a t i o n s (fig. 3a,b). Both liquids (FC 43 and FC 70) proved to be a p p r o p r i a t e and allowed r e c o r d i n g s between 20 and 35 min. Later on, the water droplets fused and thereby p r o d u c e d a short circuit. The average period length of the shuttle streaming and of the s i m u l t a n e o u s l y m e a s u r e d p o t e n t i a l was compared by Student's T-test. Both the electrical potential (x=! min 15 sec, ±SD 18 sec) and the shuttle streaming (x=1 min 14 sec, ~SD 19 sec) were of the same order of m a g n i t u d e w i t h o u t any s i g n i f i c a n t difference. In a second T-test the question of a temporal correlation b e t w e e n the points of the m i n i m u m and m a x i m u m potential values on the one hand and the change of protoplasmic streaming d i r e c t i o n on the other hand was answered. In 93% of all cases analysed such a c o r r e l a t i o n existed. The potential d i f f e r e n c e s o b t a i n e d by this m e t h o d can be caused either by rhythmic ion fluctuations b e t w e e n the slime mold and the d i s t i l l e d water or by the action of a b i o e l e c t r i c field p r o d u c e d in the mold. C o n t r o l l e d rhythmic ion fluxes across the plasma m e m b r a n e seem to be the most p r o b a b l e and i l l u m i n a t i n g e x p l a n a t i o n for the results d e l i v e r e d by the fluorinert liquid technique (c.f. the results of Ueda and Kobatake, 1977 as well as Rhea, 1966, obtained with other methods). [mY]
Fig.
3
3a
.
÷2"
i
.
.
.
.
.
.
.
.
.
.
.
.
.
.
|i
2o t[min]
PD [mY]
3b 0 .2
~~
t [rain]
Fig. 3: Two recordings of s e p a r a t e d veins in fluorinert liquid. The shuttle streaming is noted as square wave. Both r e c o r d i n g s show c o r r e l a t i o n b e t w e e n streaming and potential oscillations.
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3. M e a s u r e m e n t s w i t h intracellular m i c r o e l e c t r o d e s The addition of E D T A to the K C l - c o n t e n t of the m i c r o e l e trode allowed intracellular m e a s u r e m e n t s (between 5 and 72 min) in m a c r o p l a s m o d i a (n=ll) and d i f f e r e n t types of m i c r o p l a s m o d i a (n=35), (Gawlitta and Stockem, in preparation). Half of the obtained recordings d e m o n s t r a t e d the existence of more or less rhythmic potential oscillations (fig. 4 a,b,c,d,e) in both cells with (fig. 4 b, c,d,e) and w i t h o u t (fig. 4 a) any p r o t o p l a s m i c streaming activity. On the other hand irregular or c o m p l e t e l y smooth curves could be r e c e i v e d from macro- or microplasmodia, showing normal p r o t o p l a s m i c shuttle streaming (fig. 4 f). A r e l a t i o n s h i p b e t w e e n the extreme values of rhythmic potential alterations and the points of change of p r o t o p l a s m i c streaming d i r e c t i o n existed to some extent (fig. 4 c,d,e). The average value for the level of the m e m b r a n e potential amounted to 53.6 mY ±SD 9.9 mV and the m a i n period length in periodic recordings was c a l c u l a t e d as I min +SD 21 sec. The obtained aperiodic r e c o r d i n g s are very d i f f i c u l t to explain. Besides m e t h o d i c a l reasons (leak current along the surface of the electrode), other cell p h y s i o l o g i c a l processes (intracellular ion fluxes between d i f f e r e n t subcellular compartments) could be superimposed on the regular i o n - e x c h a n g e across the m e m b r a n e and thus c o n t r i b u t e to the i r r e g u l a r i t y of the curves. CONCLUDING RE~RKS The existence of o s c i l l a t i n g electrical activities in micro- and m a c r o p l a s m o d i a of P h y s a r u m p o l y c e p h a l u m could be d e m o n s t r a t e d with each of the three m e t h o d s tested in this investigation. This confirms the a s s u m p t i o n that rhythmic ion fluxes across the cell m e m b r a n e may play a very important role in the life cycle of this o r g a n i s m (Durham, 1974). The similar period length of the oscillating potential activity m e a s u r e d in each series of experiments (fig. 5), as well as the good a g r e e m e n t of the average value of this period length (1.21 min) with both the values for the d u r a t i o n of p r o t o p l a s m i c streaming (1.47 min, H ~ i s m a n n and W o h l f a r t h - B o t t e r m a n n , 1978) a n d radial t e n s i o m e t r i c activity (1.34 min, W o h l f a r t h Bottermann, 1977), can also be c o n s i d e r e d to support this suggestion. The fact that the o s c i l l a t i n g potential alterations could be m e a s u r e d in all types of m i c r o p l a s m o d i a - ind e p e n d e n t l y o f the existence of p r o t o p l a s m i c shuttle streaming - excludes the g e n e r a t i o n of p e r i o d i c a l ion fluxes across the cell m e m b r a n e as a c o n s e q u e n c e of the streaming activity. Regular ion exchanges could rather indicate the existence of a s u p e r i m p o s e d cytoplasmic regulatory system t r i g g e r i n g the rhythmic c o n t r a c t i o n -
Cell Biology International Reports, Vo/. 3, No. 4, 1979
327
PD [_m~l
-10 1
10
20
30
40
44
't [min}
PD [mV] -70
b -10 :
:
:
:
:
:
:
:
:
:
i
i
I
i
i
I0
i
i
i
20
i
i
i
i
i
i
i
i
,
j
. . . . . . . .
.
30
.
40
.
.1
43 t [rain}
PD [mV] -90PD
[mV] - 70
C
•
e 1
-lO
i 10
r
PD [mV] -80
16
PD [mY -90
~
-lo t [mini
1
rUU~
10
r
19
t [min]
~
d -10
-10 '
I
I
I
~
'
' ~ g
t lmin]
: : : ; i 1 1
. . . . . . . . . . . . 10
20
oi 27
t [mini
Fig. 4. Intracellular m e m b r a n e potential recordings from different types of plasmodia. a) Rhythmic potential of a sphaerical microplasmodium. b) Rhythmic potential of an amoeboid microplasmodium. c) Rhythmic potential of a fused microplasmodium. d) Rhythmic potential of a single vein of a macroplasmodium. e) Rhythmic potential of a d u m p b e l l - s ~ a p e d microplasmodium. f) Aperiodic potential of a d u m p b e l l - s h a p e d m i c r o p l a s m o dium.
Cell Biology International Reports, Vol. 3, No. 4, 1979
328
relaxation cycle of the actomyosin system (Ridgway and Durham, 1976; Kato, 1977; Ueda et al., 1978). It seems to be possible and should be proved by further experiments that ion fluxes across the cell membrane can modulate the activity of this regulatory system (e.g. by changing the period of the oscillation). Thereby they could transfer external stimuli to the cell interior, which is a necessary precondition for chemotactical reactions in general. Fig. 5: Statistical variations of the period length of electrical activity in different plasmodia
/
~oo 11o m 1
1. Measurements in fluorinert 2. M e a s u r e m e n t s
of the cell
3. Measurements
with
m>l~o sec
liquids surface
intracellular
potential microelectrodes
ACKNOWLEDGMENT We thank Mr. E. Samans for technical assistance and Prof. Dr. K.E. W o h l f a r t h - B o t t e r m a n n for providing the tensiometer equipment as well as for discussion.
REFERENCES Camp,
W.G. (1936) A method of cultivating myxomycete plasmodia. Bulletin of the Torrey Botanical Club, 63, 205. Chen, V.K.-H. (1977) Recording of m e m b r a n e potential change in Spirostomum ambiguum correlated with mechanically stimulated rapid body contraction. Abstracts of P a p e ~ r e a d at the Fifth International Congress on Protozoology, S.H. Hutner ed., 305. Daniel, J.W. and Rusch, H.F. (1961) The pure culture of Physarum p o l y c e p h a l u m on a partially soluble medium. Journal of the General Microbiology, 25, 47-59.
Cell Biology International Reports, Vol. 3, No. 4, 1979
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D'Haese, J. and Hinssen, H. (1978) Kontraktionseigenschaften von isoliertem Schleimpilzactomyosin. I. Vergleichende Untersuchungen von Fadenmodellen aus natHrlichen, rekombinierten und hybridisierten Actomyosinen von Schleimpilz und Muskel. Protoplasma, 95, 273-295. Durham, A.C.H. (1974) A unified theory of the control of actin and myosin in non muscle movements. Cell, 2, 123-136. Hatano, S. and Oosawa, F. (1966) Isolation and characterization of plasmodium actin. Biochimica et Biophysica Acta, 127, 488-498. Hato, M., Ueda, T., Kurihara, K., and Kobatake, Y. (1976) Change in zeta potential and membrane potential of slime mold Physarum polycephalum in response to chemical stimuli. Biochimica et Biophysica Acta, 426, 73-80. HHlsmann, N. and Wohlfarth-Bottermann, K,E. (1978) Spatio-temporal relationship between protoplasmic streaming and contraction activities in plasmodial veins of Physarum polycephalum. Cytobiologie, 17, 317-334, 1978. Kamiya, N. and Abe, S. (1950) Bioelectronic phenomena in the myxomycete plasmodium and their relation to protoplasmic flow. Journal of Colloid and Interface Sciences, 5, 149-163. Kamiya, N. (1959) Protoplasmic streaming. Protoplasmatologia VIII/3/a, ~Heilbrunn, L.V., F. Weber ed., Wien, Springer Verlag Berlin, Heidelberg, New York, 1-199. Kato, T. and Tonomura, Y. (1977) Uptake of calcium ions into microsomas isolated from Physarum polycephalum. Journal of Biochemistry, 81, 207-213. Kishimoto, U. (1958) Rhythmicity in the protoplasmic streaming of a slime mold Physarum polycephalum. I. A statistical analysis of the electric potential rhythm. II. Theoretical treatment of the electric potential rhythm. Journal of General Physiology, 41, 1205-1245. Olfers-Weber, R., Stockem, W. and Wohlfarth-Bottermann, E.(1976) Cytological aspects of membrane regeneration after experimental injury of amebae and acellular slime molds. Ion and enzyme electrodes in biology and medicine. M. Kessler, L.C.Clark, D.W. LHbbers, i.A. Silver, W. Simon eds., Urban & S c h w a r z e n b e r g MHnchen, Berlin, Wien, 205-216. Rhea, R.P. (1966) Microcinematographic, electron microscopic and electrophysiological studies on shuttle streaming in the slime mold Physarum polycephalum. Dynamic of fluids and plasmas. S.I. Pai et al. eds, Academic Press Inc. New York, 35-58.
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Ridgway, E.B. and Durham, A.C.H. (1976) Oscillations of calcium ion concentrations in Physarum polycephalum. Journal of Cell Biology, 69, 223-226. Ueda, T. and Kobatake, Y. (1977) Changes in membrane potential, zeta potential and chemotaxis of Physarum polycephalum in response to n-alcohols, naldehydes and n-fatty acids. Cytobiologie, 16, 16-26. Ueda, T., G~tz von Olenhusen, K., Wohlfarth-Bottermann, K.E. (1978) Reaction of the ~ n t r a c t i l e apparatus in Physarum to injected Ca--, ATP, ADP and 5'AMP. Cytobiologie, 18, 76-94. Wohlfarth-Bottermann, K.E. (1962) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung fHr die Protoplasmastr~mung. I. Elektronenmikroskopischer Nachweis und Feinstruktur. Protoplasma, 54, 514-539. Wohlfarth-Bottermann, K.E. (1963) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung fur die Protoplasmastr~mung. II. Lichtmikro~ skopische Darstellung. Protoplasma, 57, 747-761. Wohlfarth-Bottermann, K.E. (1975a) Ursachen von Zellbewegungen, cytoplasmatische Actomyosine und ihre Bedeutung fHr Protoplasmastr6mungen und Zellmotilit~t. Vortrag 1975, Halle/Saale, Sitzung der Deutschen Akademie der Naturforscher Leopoldina. Leopoldina-Mitteilungen,21, 85-128. Wohlfarth-Bottermann, K.E. (1975b) Tensiometric demonstration of endogenous oscillating contractions in plasmodia of Physarum polycephalum. Zeitschrift fHr Pflanzenphysiologie, 76, 14-27. Wohlfarth-Bottermann, K.E. (1975c) Weitreichende fibrill~re Protoplasmadifferenzierungen und ihre Bedeutung f~r die Protoplasmastr~mung. X. Die Anordnung der Aktomyosin-Fibrillen in experimentell unbeeinfluBten Protoplasma-Adern von Physarum in situ. Protistologica, XI, 19-30. Wohlfarth-Bottermann, K.E. (1977) Oscillating contractions in protoplasmic strands of Physarum: Simultaneous tensiometry of longitudinal and radial rhythms, periodicity analysis and temperature dependence. Journal of Experimental Biology, 67, 49-59. Wohlfarth-Bottermann, K.E. and G~tz von Olenhusen, K. (1977) Oscillating contractions in protoplasmic strands of P h y ~ r u m : Effects of external Ca---depletion and Ca---antagonistic drugs on intrinsic contraction automaticity. Cell Biology International Reports, l, 239-247. Received:
16th November 1 9 7 9
Accepted: 22nd January 1979
