Reactivation of cytoplasmic actomyosin in Physarum plasmodia

extracted with glycerol and dimethylsulphoxide

RENATE BELL and FRIEDHELM ACHENBACH

Institute for Cytology, University of Bonn, Ulrich-Haberland-Str. 61a, D-5300 Bonn 1, Federal Republic of Germany

Summary

Thin-spread plasmodia of Physarum were sub-jected to extraction procedures using 50%glycerol or DMSO (dimethylsulphoxide) fol-lowed by labelling of actin -with fluorescentphallotoxins.

During the reactivation of the actomyosin sys-tem by 2 mM-MgATP fluorescent actin fibres con-tract isotonically, which results in numerousfluorescent 'contraction beads'.

After short-term extraction 1 mM-Ca2+ hasan inhibitory effect on the reactivation. Thiscalcium sensitivity is abolished after long-termextraction with glycerol.

Calcium at 10 mM irreversibly inhibits reacti-vation, irrespective of the duration of extraction.The inhibitory effect of 10 mM-calcium is pre-vented by phallotoxin labelling prior to incu-bation in Ca2+.

The DMSO model shows an improvement instructural preservation when compared with theglycerol models. However, reactivation is in-hibited by prolonged treatment with DMSO.

Key words: Physarum polycephalum, reactivation ofcytoplasmic actomyosin, calcium, fluorescence microscopy.

Introduction

The plasmodial stage of Physarum polycephalumserves as a model in the study of the physiologicalconditions of cytoplasmic actomyosin function in situ(Kamiya, 1959; Kamiya & Kuroda, 1965; Wohlfarth-Bottermann, 1975). The functional regulation of theactin-myosin interaction has been subjected tointensive investigation by different approaches,in Physarum (Gotz von Olenhusen & Wohlfarth-Bottermann, 1979; Kohama & Shimmen, 1985;Kohama et al. 1985; Maruta et al. 1983), and in othernon-muscle systems (e.g. see Holzapfelei al. 1983). Inmost cell types a calcium/calmodulin/myosin lightchain-kinase-dependent phosphorylation of myosinseems to be the regulating mechanism (Masuda et al.1984).

Cell-free models as tools in the study of the contrac-tile apparatus in situ were introduced in the 1940s(Szent-Gyorgyi, 1949). Pitfalls and improvements incell-free models of Physarum have been reviewedrecently (Wohlfarth-Bottermann, 1985). The follow-ing types of models are available. (1) Glycerol modelof endoplasmic drops (Achenbach, 1985; Achenbach& Wohlfarth-Bottermann, 1986a). (2) 'Cryosection

Journal of Cell Science 87, 231-239 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

model' (Pies & Wohlfarth-Bottermann, 1984, 1986).(3) DMSO (dimethylsulphoxide) model of endoplas-mic drops and plasmodial strands (Achenbach &Wohlfarth-Bottermann, 19866). (4) Different deter-gent-permeabilized models (Ogihara, 1982; Yoshi-moto et al. 1981; Yoshimoto & Kamiya, 1984).

We have prepared glycerol and DMSO models ofthin-spread plasmodia using fluorescent phallotoxinsas a stain to visualize plasmodial actin fibrils and theircharacteristic disintegration into 'beaded chains'during reactivation by MgATP, as recently describedfor the 'cryosection model' (Pies & Wohlfarth-Botter-mann, 1984, 1986).

The aim of the present study was to follow directlychanges in the structure of actomyosin fibrils duringreactivation and to elucidate the role of Caz+ in theregulation of contraction.

Materials and methods

Preparation of plasmodiaThin-spread plasmodia of Physarum polycephalum (strainATCC 44912) were prepared according to Kamiya &Kuroda (1965) with the modification that plasmodia were

231

Table 1. Summary of preparation procedures and composition of solutions used in six different experimentalseries (A—F)

Extraction

Washing

Labelling

Washing

Reactivation

For details

A

SES5, 10, 15,30min;1, 15-20d

SWS5 min—3 h

SWS +RHPHN orRHPHD30 min

SWS5 min—1 h

SRS5 min

of solutions see

B

SES5, 10, 15,30 min;1, 15-20d

SWS +0-lmM-Ca2+ or1 mM-Caz+ or10mM-Ca2+

5 min—3 h

SWS+ 0-1, 1 or10mM-Ca2+ +RHPHN30-60min

SWS + 0-1, 1 or10mM-Ca2+

5 min—1 h

SRS + 0 1 , 1 or10mM-Ca2+

5 min

Materials and methods.

C

SES5, 30min;1, 15-20d

SWS10 min

SWS +RHPHN30 min

SWS+ 0-1, 1 or10mM-Ca2+

10-60 min

SRS + 0-1, 1 or10mM-Ca2+

5 min

d, days.

D

SES30 min,3h, l -7d

SWS +10mM-Ca2+

30min, l -4hSWS +lOmM-EDTA15 minor SWS +lOrrun-EGTAlh , SWS 15 min

SWS +RHPHN orRHPHD30 min

SWS10 min

SRS5 min

E

25, 40 or50%DMSO,lOmM-EGTA,lOmM-Tris-HCl5, 30min, 2h, Id

SWS +lOmM-EGTA15 minSWS15 min

SWS +RHPHN orRHPHD30 min

SWS5 min

SRS5 min

F

40%DMSOlOmM-EGTAlOmM-Tns-HCl5, 15, 30min, 2h

SWS +lOmM-EGTA15minSWS15 min

SWS +RHPHN orRHPHD30 min

SWS + 0-1, 1 or10mM-Ca2+

30-120 min

SRS+ 0 1 or1 mM-Ca2+

5 min

also grown at 15°C and without covering with a cellophanemembrane and agar 9heet.

SolutionsThe translucent plasmodia were transferred into a cold(5°C) standard extraction solution (SES) containing 50%(v/v) glycerol, lOmM-EDTA and 10mM-Tris-HCl. Inanother series the plasmodia were extracted at room tem-perature in 25%, 40% or 50% DMSO, containing 10 raM-EGTA and lOmM-Tris-HC1 for at least 5 min and up to24 h (for further details see Table 1,E,F). The standardwashing solution (SWS) was 30mM-KCl, 1 mM-MgCl2 and10mM-Tris-HCl; the standard reactivation solution (SRS)contained 30mM-KCl, 2mM-MgCl2> lOmM-TrisHCl and2mM-ATP. Further treatment with different washing sol-utions, the phallotoxin staining and the reactivation sol-utions were applied at room temperature, as listed inTable 1. All solutions were adjusted to pH6-9.

Ar-ethylmaleimide (NEM, 5mM) or 1 mM-p-chloromer-curibenzoic acid (PCMB) in SWS for 15 min were appliedprior to fluorescent staining. The reversal of PCMB inhi-bition was performed by washing in lOmM-L-cysteine for1-4 h.

CytochemistryIn most cases, filamentous actin was stained with RHPHN(rhodamine-phalloin, a gift from Professor Th. Wieland,Heidelberg), or with RHPHD (tetramethylrhodaminyl-

phalloidin, Molecular Probes, Inc., Junction City, Oregon).The methanolic stock solutions (3-3yM) were diluted toa final concentration of 06fiM after evaporation of themethanol. Staining was performed with 10—20^1 of thefluorescent probe per plasmodium for 30 min.

Light microscopy and video techniquesThin-spread plasmodia growing on agar sheets were trans-ferred upside-down to a coverslip to be viewed with aninverted microscope (Zeiss IM35 equipped with phase-contrast, 546 nm fluorescence excitation and Plan-Neofluaroptics). Reactivation solution was applied immediately onthe thin agar layer covering the model, reaching it within1-2 min by diffusion.

Each plasmodium was divided into two halves, one beingused as a control (SWS and SRS, Caz+-free) and the otherfor the test. A reactivation was judged to be positive ifruptured fibrils or actomyosin sheets could be seen in severalregions of the models.

For fluorescence micrography Kodak Tri-X pan film wasprocessed to ASA 1600 in Diafine.

A K5B television compact camera (Siemens, Darmstadt,FRG) coupled with an integral image intensifier for fluor-escent signal enhancement served to store time-lapse videosequences on a Sony U-matic recorder VO-5800PS). Imageprocessing was done using an IBAS II computer (Zeiss/Kontron).

232 R. Bell and F. Achenbach

Results

Extraction

After glycerol extraction the cytoplasm has a vacuolarappearance, and the organelles are damaged consider-ably. However, actomyosin fibrils could still be re-activated.

The shortest extraction time that reliably producedplasmodia devoid of living portions of cytoplasm was5 min. Hence, to avoid possible errors, we ensuredthat the extraction times exceeded 5 min (Wohlfarth-Bottermann, 1985).

Reactivation

The reactivation experiments were judged to be posi-tive if the fibrils disintegrated into 'beaded chains'(Fig. 1) after addition of MgATP. If only corticalactomyosin sheets and/or small portions of singlefibrils ruptured, the reactivation was judged to beweak.

The frontal zone in Fig. IB exhibits an incompletecontraction of fibrils, probably due to an onset ofinhibition by irradiation (cf. Pies & Wohlfarth-Botter-mann, 1984, 1986). Fig. 1D,F shows fully contractedactomyosin polygons and fibrils, resulting in densecontraction beads.

Inhibitor experiments

Incubation of the models in a washing solution con-taining 5 mM-A'-ethylmaleimide for 15 min prior tolabelling led to an irreversible inhibition of reacti-vation. Inhibition was also obtained with 1 mM-PCMB. The inhibitory effect of PCMB could bereversed by further incubation in an excess of L-cysteine (10 mM in SWS for 1-3 h). Reactivation couldbe prevented completely by omitting Mg2"1" in thecontraction solution.

Influence of Ca2+

To avoid errors due to individual differences inplasmodia, each plasmodium was divided into twoportions, one being used as a control (Table 1A).

In Fig. 2 the dependence of reactivation on theduration of extraction is summarized for the controls,i.e. reactivation in the absence of Ca2+ (Table 1A).Columns marked by the extraction times represent thepercentage of models reactivated after 5 min (whitecolumns) and 3 h (black columns) of washing. Theability to reactivate was independent of the durationof extraction. Thorough washing improved the re-activation.

Fig. 3 shows an attempt to evaluate reactivationexperiments qualitatively by scaling the degree ofreactivation in individual models from 0 (no reacti-vation) to 2 (full reactivation). The evaluation corrob-orates results summarized in Fig. 2, i.e. reactivation is

independent of the duration of extraction, and it isimproved by thorough washing.

The number of control models reactivated in eachexperimental series showed individual differences de-pending on several uncertainties. Therefore this valuewas normalized to 100%, and the relative number ofreactivations in the calcium experiments (Figs 4, 5)was calculated accordingly.

Results concerning the effect of calcium on thereactivation of glycerol models are summarized inFig. 4. Ca2+ was present before, during and afterfluorescent staining (Table IB). Ca2+ (lOniM) com-pletely prevented reactivation and almost full reacti-vation was achieved in the presence of 0-1 mM-Ca2+ orless; in both cases these effects were independent ofthe duration of extraction. However, with 1 mM-Ca2+

an inhibitory effect was observed if plasmodia wereextracted for less than 30 min, while reactivationoccurred after long-term extraction.

In the presence of 1 mM-Ca2+ prolonged washing ofshort-term extracted specimens did not result inreactivation.

To show whether incubation in 10 mM-Ca2+ prior tofluorescent labelling irreversibly blocks contraction,lOmM-EDTA or EGTA was used to remove thecalcium before staining and reactivation (Table ID).In these experiments the plasmodia still containedfibrils but none contracted.

If phallotoxin staining was performed prior toreactivation at different pCa values (Table 1C), theinhibitory effect of 10mM-Ca2+ was restricted toshort-term extraction (Fig. 5). In contrast to modelsstained before treatment with 10mM-Ca2+, the fluor-escent labelling was weak in plasmodia in whichstaining followed the Ca2+ treatment. Calcium (1 mM)also tended to prevent reactivation after short-termextraction, whereas 0-lmM-Ca2+ did not decisivelyimpair reactivation, regardless of the extraction time.The Ca2+ sensitivity is abolished after long-termextraction.

When 10mM-Ca2+ was completely removed bylOmM-EGTA in plasmodia not capable of beingreactivated after short-term extraction, reactivationoccurred in about 30 % of the models.

Contraction was not inhibited in preparations inwhich 10mM-Ca2+ was replaced by 10mM-Mg2+.

Registration of the isotonic contraction by video-enhanced microscopy

With the help of time-lapse video-enhanced mi-croscopy the mode of contraction of the actomyosinfibrils could be studied in more detail. Immediately

Reactivation of cell-free models o/Physarum 233

after addition of MgATP movement could be seen as

the initial reaction: plasmodial strands seemed to

cramp and shift before the fibrils began to contract,

i.e. to rupture. In about one third of the plasmodia the

diameter of the strands visibly decreased by about

2-10% of the initial value.

Fig. 1. Reactivation of glycerol models of thin-spread plasmodia made visible by fluorescence microscopy following stainingwith fluorescent rhodamine-phalloin. A,B. Plasmodial frontal zone before (A) and after (B) reactivation. Note that in thiscase reactivation is incomplete. C,D. Plasmodial frontal zone before (C) and after (D) complete reactivation, i.e.disintegration of the fibrils into rows of beads. E,F. Plasmodial strand before (E) and after (F) reactivation. Note the slightdecrease in diameter of the strand. Bar, 50 fim.

234 R. Bell and F. Achenbach

* 50

5min 15min Id 15-20 d

Fig. 2. Evaluation of the influence of extraction time (t) onthe reactivation of glycerol models. Ordinate: percentage ofplasmodia reactivated (r) after 5 min (white columns) and3h washing (black columns), d, days.

The mode of contraction varied depending on thefibrillar pattern. Actomyosin fibrils of the frontal zoneoften contracted to form compact spots without disin-tegration. In some cases thick bundles of fibrilssurrounding the endoplasmic channel first disinte-grated into larger segments and then single fibrilsbegan to rupture.

Disintegration is characterized by the formation of arow of fluorescent beads, resulting in the appearanceof beaded chains after complete contraction. At theonset of contraction the sites of dissection are markedby a reduced fluorescence, probably indicating theonset of disintegration (Fig. 6). At higher magnifi-cation, the 'beads' within one former fibril were similarin size; on the other hand, different fibrils producedbeads of different size, apparently depending on theinitial diameter of the fibril.

DMSO extraction

Reactivation of plasmodia extracted with 25-50 %DMSO was similar to that in the glycerol models(Table IE). However, the structural preservation wasimproved. Long-term extraction led to the inhibitionof reactivation: Fig. 7 demonstrates that increasing

- 1 -

5 min 15 min 15-20d

Fig. 3. Evaluation of contraction intensity (i) after scalingfrom 0 (no reactivation) to 1 (weak reactivation) and 2(strong reactivation) depending on the extraction time (t).White columns: 5 min washing; black columns: 3hwashing. Prolonged washing improves reactivation of themodels.

100

* 50

5 10 15 30 min Id 15-20d

Fig. 4. Calcium dependence of reactivation in glycerolmodels treated with Ca2+ before labelling. Abscissa:extraction time (t); ordinate: relative number of plasmodiareactivated under different calcium conditions (r) ascompared to controls (=100%), i.e. models reactivated atzero calcium. ( • ) 0-1 mM-Ca2+; (T ) 1 mM-Ca2+;( • ) 10mM-Ca2+.

concentrations of DMSO and prolonged extractiontime inhibited contraction of the models.

Calcium concentrations of 0-1 mM or less supportedreactivation after short-term extraction in 40%DMSO (Fig. 8). Ca2+ at 1 mM inhibited reactivationirrespective of the duration of extraction.

Discussion

The reactivation of cytoplasmic actomyosin fibrils incell-free models results in disintegration, visible in thefluorescence microscope after specific actin stainingwith fluorescent phallotoxins. This type of behaviourwas first described by Naib-Majani et al. (1984) insandwiched plasmodia and later by Pies & Wohlfarth-Bottermann (1984, 1986) in cryosections of plas-modial strands. It was shown by light and electronmicroscopy that this phenomenon is a result of the

100

* 50

5 min 30 min Id 15-20d

Fig. 5. Experiments and evaluation as in Fig. 4, butcalcium treatment was performed after phallotoxinlabelling. Symbols and abbreviations as in Fig. 4.

Reactivation of cell-free models o/Physarum 235

complete isotonic contraction of actomyosin fibrik(Pies & Wohlfarth-Bottermann, 1984).

The calcium sensitivity of plasmodial models issimilar to the cryosection model (Pies & Wohlfarth-Bottermann, 1986), i.e. reactivation of plasmodial

actomyosin is increasingly inhibited by increasingconcentrations of Ca2+ (pCa 3-4). Also in Chara, theblocking effect of Caz+ on cytoplasmic streaming liesin the same range (Hayama et al. 1979). Glycerol andDMSO models of endoplasmic drops and plasmodial

Fig. 6. Course of reactivation analysed by time-lapse, video-enhanced fluorescence microscopy. In two individual fibrils thesites of distintegration are marked by black and white arrowheads. Numbers indicate seconds after addition of ATP. Bar,50 ^m.

236 R. Bell and F. Achenbach

25%

40%

50%

5 min

+

+

+

30 min

+

+/±

+

2 h

+

-

18 h

+

-

-

Fig. 7. Evaluation of experiments following extraction in25, 40 and 50% DMSO for different periods of time. Notethat the ability to reactivate increases with decreasingDMSO concentration (c) and decreasing extraction time(/). +, strong reactivation; ± , weak reactivation; —, noreactivation.

0.1mM

1mM

0Control

5 min

+

+

15 min

+

-

+

30 min

-

2 h

-

*/t/-

Fig. 8. Calcium dependence of reactivation of DMSOmodels. At 0-l mM-Ca2+ and less, the models arereactivated. 1 mM-Ca2+ totally blocks reactivation. If theextraction time exceeds 30 min, an increasing impairmentof reactivation is observed at zero Ca2+ concentration.

strands are reactivated at calcium concentrations of10~7M and below, whereas 10~5M-Ca2+ inhibits reac-tivation (Achenbach, 1985; Achenbach & Wohlfarth-Bottermann, 1986a,6). This discrepancy in the ordersof magnitude cannot be explained at present. How-ever, it seems likely that in models of thin-spreadplasmodia or cryosections extraction occurs morerapidly than in endoplasmic drops, i.e. calcium sensi-tivity is more rapidly removed. In DMSO models ofthin-spread plasmodia the duration of extractionshould be below 30 min, otherwise reactivation isincreasingly inhibited, i.e. a pronounced calciumsensitivity of reactivation can be observed only aftershort-term extraction (Fig. 8).

Early reports concerning the calcium control ofPhysarum actin-myosin interaction favoured the ideaof a muscle-like situation (Hatano, 1970; Ogihara,1982; Ueda et al. 1978; Wohlfarth-Bottermann et al.1983).

Inhibitory calcium regulation of actin-myosininteraction has been demonstrated by several authorsusing different approaches (Achenbach & Wohlfarth-Bottermann, 1986a,6; Kohama & Kendrick-Jones,1986; Ogihara et al. 1983; Pies & Wohlfarth-Botter-mann, 1984, 1986; Yoshimoto et al. 1981; Yoshimoto& Kamiya, 1984). Myosin light chain 2 seems to be the

calcium-sensitive regulatory factor (Kessler et al.1980; Kessler & Dolberg, 1986; Kohama & Ebashi,1986). Also, in other cells an inhibitory effect ofcalcium on the cytoplasmic actin-myosin interactionwas reported (Kuroda & Sonobe, 1981; Yoshimoto &Hiramoto, 1985). In addition to the possible role ofcalcium in myosin-linked regulation, its interferencewith actin-linked regulatory components has to beconsidered (Hatano, 1986; Hinssen, 1981). Kohamaet al. (1985) discussed the involvement of a cyto-plasmic light chain 2 in binding to actin in a calcium-sensitive inhibitory manner.

The fact that plasmodial actin could be fluor-escently labelled in the models, even after theirreactivation was completed, indicates the maintenanceof F-actin in the 'contraction beads', i.e. actin in ourmodels probably does not depolymerize during reacti-vation. In addition, the role of actin-severing proteins(e.g. fragmin) is obscure both in the models and inliving cells. However, the calcium-dependent regu-lation of actin polymerization is consistent with theresults.

Ca2+ at 10 mM seems to interfere with phallotoxinstaining, indicating that at least part of the filamentousactin is depolymerized by this high concentration(but not by magnesium). In addition, reactivation isirreversibly inhibited by 10mM-Ca2+. This cor-roborates results obtained with Chara internodal cells,in which cytoplasmic streaming, once inhibited byhigh Ca + , could not fully recover after removal ofCa2+ with EGTA (Hayama et al. 1979). The inhibi-tory effect may be caused by the removal and/orinactivation of some regulatory component compar-able to the 'desensitizing' effect of EDTA on theregulatory light chains of scallop myosin (Vale et al.1984).

The impression gained from time-lapse investi-gations of the reactivation process is that immediatelyafter addition of MgATP a rapid, low-amplitudeisotonic contraction of the entire model occurs, fol-lowed by the slower process of disintegration of fibrils.The disintegration may indicate intrinsic sarcomere-like repeated substructures of the fibrils (perhapssimilar to the situation in tissue-culture cells; Lang-anger et al. 1984, 1986) or regions of differentmechanical stability enabling rupture of weaker areas.The distance apart of fluorescent beads varies between0-6 and 10/xm. The smallest values approximate theorder of magnitude of the repetitive distances betweenar-actinin units in stress fibres of tissue-culture cells(Langanger et al. 1984, 1986).

The authors thank Professor Dr K. E. Wohlfarth-Botter-mann, Drs K. Kohama (Tokyo), N. Pies, W. Naib-Majaniand D. Gassner (Bonn) for helpful discussions, Dr J.Kukulies (Bonn) for help with image processing and DrR. L. Snipes (Giessen) for reading the manuscript. The

Reactivation of cell-free models of Physarum 237

work was supported by the Deutsche Forschungsgemein-schaft (Wo 20/22-2).

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(Received 30 September 1986-Accepted 20 November1986)

Reactivation of cell-free models of Physarum 239