Physical and Chemical Basis of Cytoplasmic Streaming

Annual Reviews www.annualreviews.org/aronline

PHYSICAL AND CHEMICAL BASIS OF CYTOPLASMIC ~7710 STREAMING

Nobur6 Kamiya

Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444 Japan

CONTENTS

INTRODUCTION...... 206 SHUTI’LE STREAMINGIN THE MYXOMYCETEPLASMODIUM ...... 207 General...... 207 ContractileProperties of the PlasmodialStrand ...... 208 Activationcaused by stretching ...... 208 Activationcaused by loading ...... 209 Synchronizationof local ,hythms ...... 209 ContractileProteins ...... 210 Plasmodiumactomyosin ...... 210 Plusmodiummyosin ...... 210 Plusmodiumactin...... 211

by STEWARD OBSERVATORY on 04/23/06. For personal use only. Tension Production of Reconstituted Actomyosin Threads from Physarum...... 212 Regulationof Movement ...... 213 Therole of Ca ~...... 213 Therole of ATP ...... 214 StructuralBasis of MotiliO~...... 216 Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org Contraction-relaxationcycleand actln transformations...... 216 FibrillogenesiS...... 217 Birefringence...... 217 Summary...... 218 ROTATIONALSTREAMING IN CHARACEANCELLS ...... 219 General...... 219 TheSite of MotiveForce Generation ...... 219 Subcortical Filaments, Their Identification as Actin, and Their Indispensability forStreaming ...... 220 NitellaMyosin and its Localizationin the Cell ...... 221 Motility of Fibrils and Organellesin Isolated CytoplasmicDroplets ...... 222 Rotationof chloroplasts ...... 222 Motilefibrils ...... 223 2O5 0066-4294/81/0601-0205501.00 Annual Reviews www.annualreviews.org/aronline

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DemembranatedModelSystems ...... 224 Removaloftonoplast byuacuolar perfu~ion ...... 224 Demembronatedcytoplasmicdroplets ...... 224 Activemovement in vitro of cytoplasmicflbeCls ...... 225 Measurementof theMoti~e Force ...... 225 Centrifugationmethod...... 226 Perfusionmetkod ...... 226 Methodoflateral compassion ...... 227 MolecularMechanism of Rotational Streaming ...... 228 CONCLUDINGREMARKS...... 229

INTRODUCTION

Twodecades have elapsed since this author wrote a review on cytoplasmic streaming for VolumeI 1 of this series (72). As is the case with other types of cell motility, research on cytoplasmic streaming has madegreat strides during this period--including isolating proteins related to streaming, eluci- dating its ultrastructural background, and developing new effective methods for studying functional aspects of cytoplasmic streaming. There are a numberof reviews and symposia reports dealing with various aspects ofnonmuscularcell motility including cytoplasmic streaming (8, 10, 11, 23, 28, 31, 36, 48, 53, 59, 60, 62, 99, 100, 134, 136, 139, 15o, 151, 159). They will provide readers with more comprehensive information on the subject. In this report, I shall limit myconsideration to someselected topics in cytoplasmic streaming, focusing on its mechanismat the cellular and molecular levels. Generally speaking, minute structural shifts in the cytoplasm maybe a wide oecurrance in living cells, but they will not necessarily develop into significant movementunless they are coordinated. In a variety of cells, cytoplasmic particles are knownto make sudden excursions over distances too extensive to be accounted for as Brownianmotion (136). Such motions, called "saltatory movements,"were described long ago in plant literature

by STEWARD OBSERVATORY on 04/23/06. For personal use only. as "Glitchbewegung" or "Digressionsbewegung" (of 71, 73). The movement of the particles is erratic and haphazard, yet it is not totally devoid of directional control as it would be in Brownianmotion. Accordingto the

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org degree of orderliness, intraeellular streaming manifests various patterns (71, 73). Cytoplasmic movementsexhibited by eukaryotic cells maybe classified into two major groups with respect to the proteins involved, i.e. the actin- myosin system and the tubulin-dynein system. Cytoplasmic streaming be- longs mostly to the first group. Possible roles for the tubulin-dynein system in cytoplasmic streaming have yet to be investigated. Fromthe phenomeno- logical point of view, it is customaryto classify the streaming of cytoplasm at the visual level into two major categories. One is the streaming closely associated with changes in cell form. This type of movementis usually Annual Reviews www.annualreviews.org/aronline

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referred to as amoeboid movementand is represented by the amoeba, acellnlar slime molds, and manyother systems. The other is the streaming not dependentupon changesin the external cell shape, as in most plant cells or dermatoplasts. In the following sections, I shall discuss the two best studied cases as representative modelsof cytoplasmic streaming. Oneis shuttle streaming in myxomyceteplasmodia of the amoeboid type, and the other is rotational streaming in eharacean cells of the other type. It is necessary to describe them separately because we still do not knowat what organizational level these two major categories of streaming share a commonmechanism.

SHUTTLE STREAMING IN THE MYXOMYCETE PLASMODIUM General The plasmodium of myxomycetes, especially that of Physarum polycephalum, is classic material in whichthe physiology, biochemistry, biophysics, and ultrastructure of cytoplasmic streaming have been investigated most extensively (20, 35, 36, 71, 99, 133). The myxomyceteplasmodium shows various characteristic features in its cytoplasmic streaming. The rate of flow, as well as the amountof cytoplasm carded along with the streaming, is exceedingly great comparedwith ordi- nary cytoplasmic streaming in plant calls (71). Moreover, the direction streaming alternates according to a rhythmic pattern. There is good evidence to show that the flow of endoplasmis caused passively by a local difference in the intraplasmodial pressure (83). This differential pressure the motive force responsible for the streaming. It can be measuredby the so-called double-chambermethod, in which counterpressure just sutfieient to keep the endoplasm immobileis applied (70, 71).

by STEWARD OBSERVATORY on 04/23/06. For personal use only. The waves representing spontaneous changes in the motive force are sometimesvery regular, but the amplitude of the waves often increases and decreases like beat waves. In someother cases, a peculiar wavepattern is

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org repeated over several waves. These waves can be reeonstrneted closely enough with only a few overlapping sine waves of appropriate periods, amplitudes and phases. This fact is interpreted as showingthat physiological rhythms with different periods and amplitudes can simultaneously coex- ist in a single plasmodium(70, 71). A variety of physical or chemical agents in the production of the motive force have been investigated (71). Recently, extensive analysis of tactic movementsof the slime mold was made by Kobatake and his associates (47, 153-155). Threshold concentrations for the recognition of attractants (glu- cose, galactose, phosphates, pyrophosphates, ATP, cAMP)and of repel- Annual Reviews www.annualreviews.org/aronline

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lents (such as various inorganic salts, sucrose, fructose) were thus determined (155). It has been suggested that recognition of chemical sub- stances is.caused by a change in membranestructure which is transmitted to the motile systems of the plasmodium.Motive force production is closely related to bioelectric potential change(79) as well as anaerobic metabolism (71, 138).

Contractile Properties of the Plasmodial Strand Since the streaming of the endoplasmis a pressure flow, and the internal hydrostatic pressure of the plasmodiumis thought to be produced by contraction of the ectoplasm, the contractile force of the ectoplasm per se should serve as a basis for analyzing cytoplasmic streaming in this organism. Contractility was often assumedto be involved in a variety of movements of nonmuscular cells. Nevertheless, contractile force was not measureddirectly and precisely in motile systems other than muscles until it was measured in an excised segment of a plasmodial strand (75, 76, 78, 80, 88, 89, 148, 170, 171). The strand forming the network of the plasmodiumis actually a tube or vein with a wall of ectoplasmic gel. The endoplasmflows inside the ectoplasmic gel wall. Thoughendoplasm and ectoplasm are mutually interconverti- ble, it is the ectoplasmic gel structure that is mainly responsible for the dynamicactivities of the strand (152). The absolute contractile force of the ectoplasm can be measured in this case as a unidirectional force. The maximal contractile force so far measured is 180 gcm-2 (78). Dynamic activities of the plasmodial strand can be expressed in terms of spontaneous changes in tension while the length of the strand is kept constant (isometric contraction), or in terms of spontaneous changes in length keeping constant the tension applied to the strand (isotonic contraction).

by STEWARD OBSERVATORY on 04/23/06. For personal use only. ACTIVATIONCAUSED BY STRETCHINGOne of the outstanding characteristics of the plasmodial strand is its response to stretching. Whena strand segment is stretched, say by 10-20%,the tension and amplitude of

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org the waveincrease immediately while the waveperiod remains constant (76, ¯ 80, 176). There is no shift in phase (78, 148, 176). Wohlfarth-Bottermarm and his co-workers (2, 101), however, report a phase shift whenthe strand is stretched as muchas 50%. After stretching, the wavetrain movesdown- ward rather rapidly at first and less rapidly afterwards, showing tension relaxation under isometric conditions. The increase in amplitude of the tension waves of the plasmodial strand by stretching, and its subsequent decrease, are comparable to those shownby the motive force waves of the surface plasmodiuminflated with endoplasm by external pressure (74). Annual Reviews www.annualreviews.org/aronline

CYTOPLASMICSTREAMING 209

ACTIVATIONCAUSED BY LOADINGUnder isotonic conditions, the situation is somewhatdifferent from the above. If the load is increased, the amplitude of the waves increases also. Whenthe load is decreased, the amplitude decreases correspondingly. With constant tension levels, the cyclic contraction waves do not tend to decrease their amplitude. This result is in contrast to isometric contraction wavesafter stretching. The increase of the amplitude of the isotonic contraction waves under greater tension is not accompaniedby an increase in period length. This result indicates that the speed of both contraction and relaxation increases rather than decreases under higher tension (76, 80). In other words, the contracilc capacity of plasmodial strand segmentis activated by the tension applied externally. In order to understand this remarkable phenomenon,we have to postulate the presence of some regulatory mechanism by which the plasmodium can "sense" the tension change first, and then control the force output to correspond with the amount of load.

SYNCHRONIZATIONOF LOCALRHYTHMS A strand segment shows no significant rhythmic activities soon after it is excised from the mother plasmodium.It starts rhythmic contraction locally after 10-20 min. Thirty minutes later, small local rhythms becomegradually synchronized to form a unified larger rhythm (175). Takeuchi & Yoneda(141) reported that whenindividual strand segments of P/~ysarumplasmodium having different contraction-relaxation periods were connected by way of a plasmodial mass, the cycles of the two segments becamesynchronized. To clarify the possible role of the streaming endoplasm as the information carder for synchronization, Yoshimoto& Kamiya (175) set a single segment ofplasmodial strand in a double chamberin such a way that the two halves of the segment were suspended in different compartments of the chamber. The strand penetrating the central septum

by STEWARD OBSERVATORY on 04/23/06. For personal use only. of the double chamber thus took an inverted U-shape. Whenthe shuttle streaming of the endoplasmoccurred freely between the two halves of the strand, they contracted and relaxed in synchrony. But if balancing counterpressure was applied to one of the two compartmentsto keep the endoplas- Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org mic flow in the strand near the septum of the chamberat a standstill, then the contraction-relaxation rhythms of the two halves movedout of phase with each other. Whenthe endoplasmwas allowed to stream freely again, the synchrony of their cyclic contractions was reestablished. Thus Yo- shimoto & Kamiya (177) concluded that endoplasm must carry some factor(s) whichcoordinates the period and phase of the contraction-relaxation cycle. It did not control the amplitude of the oscillation. In other words, information necessary for unifying the phases of the local contraction- Annual Reviews www.annualreviews.org/aronline

210 KAMIYA

relaxation cycle is transmitted neither by electric signal nor by direct mechanical tension of the endoplasm. The nature of the factor(s) carried the endoplasmis still unknown.

Contractile Proteins

PLASMODIUMACTOMYOSIN Cytoplasmic actomyosin is now known to bc presentthroughout cukaryotic cells (I 34,160-162). It is responsiblefor a varietyof cellmovements. The presence of an actomyosin(myosin B)-likc proteincomplex in the myxomyceteplasmodium was shown by A_dad Locwy(108) as earlyas 1952.This study is a pioneeringwork on contractile proteinsin nonmusclcsystems. Since then, the biochemicalproperties of contractilcproteins in theseorganisms have bccn studied extensively. Pro- teinssimilar to musclemyosin and actin wcrc subscqucntly extracted from theplasmodium and purified separately. Fortunately, there arc comprehen- sivearticles and symposia reports in thisarea (3, 35, 37, 42, 116); hence shalldescribe the matter only briefly here. Likemuscle actomyosin, plasmodium actomyosin shows supcrprccipita- tionat lowsalt concentrations andviscosity drop at highconcentrations on additionof Mg2+-ATP(35, 116, 126). ATPasc activities of plasmodium actomyosinarc basicallysimilar to thoseof muscleactomyosin. Plas- modiumactomyosin having Ca2+-sensitivity hasalso been isolated (92, 93, 117). Superprecipitation of the protein is observed only in the presence of a mieromolar order of free Ca2+; the Mg2+-ATPaseis activated two- to sixfold by 1 btM free Ca2+. This Ca2+ sensitivity is thought to be caused by the presence of regulatory proteins as in skeletal muscle. A noteworthy characteristic of Physarumactomyosin, which is not shared with muscle actomyosin, is that superprecipitation is reversible, i.e. it can be repeated several times on addition of ATP(111). by STEWARD OBSERVATORY on 04/23/06. For personal use only. PLASMODIUMMYOSIN The moleculeof plasmodiummyosin has a rod- likestructure with a globularhead on oneend just like that of striated

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org myosin(43). PIasmodium myosin can combinewith plasmodium F-actin, andmusclc F-actin as well,to formactomyosin-likc complcxcs (35, 45). Thc molecularweight of theheavy chain is 225,000daltons as determinedby SDS gel electrophoresis.Plasmodinm myosin has ATPase activity similar to thatof myosinfrom muscle, but in contrastto musclemyosin, plasmodiummyosin is solubleat neutralpH andlow saltconcentrations, in- cludingphysiological concentration (0.03 M KCI).Hinssen (56), Hinssen & D’Haese (57), Nachmias (114, 115), and D’Haese & Hinssen (26) strated the capacity of Physarummyosin to self-assemble into thick, bipolar aggregates or long filaments. In comparison with muscle myosin, plas- Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 211

modiummyosin can form stable thick filaments only under a strictlydefined rangeof conditionswith respect to ionicstrength, ATP concentration, and pH.Though divalent cations arc not absolutely necessary, filament forma- tionis improvedby Mg2+ concentrationsup to 2 mM and by Ca2+ up to 0.5raM. Propcrti~ so farknown for P/~ysarum myosin arc listed in a table withthe references by Nachmias(I 16).

PLASMODIUMACTIN Plasmodium actin was isolated by Hatano & Oosawa(39, 40) from Physarumby using its specific binding to muscle myosin; it was purified by salting out with ammoniumsulfate. This was the first time actin was isolated from nonmusclecells. Since then actin has been isolated from manynonmuscle cells. Actin is nowknown to be a ubiquitous and commonprotein in eukaryotic cells. The physical and chemical properties of actins from various nonmusclcsources including Physarumare all similar to those of muscle actin. The protein is in a monomericstate in a salt-free solution, giving a single sedimentationcoefficient of about 3.5 S (4, 40). The molecular weights of actins from muscle and plasmodiumare both about 42,000 in the SDSgel electrophoresis system. Analysis of amino acid composition of these two types of actins has indicated someminor dispari- ties (42). The amino acid sequence of Pl~ysarumactin has recently been determined and shows a difference from mammalianT-cytoplasmic actin in only 4%of its primary structure (157). The difference in amino acid sequence between Physarum actin and rabbit skeletal muscle actin was determined to be 8%. On addition of salts such as KC1, actin monomers polymerizc into F-actin with concomitant hydrolysis of ATP. Electron micrographs showed that this polymer takes a form of helical filament identical to those of F-actin from muscle. PlasmodiumF-actin also forms a complex with heavy mcromyosin (HMM)from muscle to make an arrowhead structure (13, 118, 122).

by STEWARD OBSERVATORY on 04/23/06. For personal use only. The actin preparation obtained by Hatano and his collaborators (35, 46) formed an unusual polymer termed "Mg-polymcr" with 0.1 to 2.0 mM MgCl2.Although the sedimentation coefficient of this polymer is about the same as that of F-actin, Mg-polymerhas a muchlower viscosity, less flow Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org birefringcnce, and appears as a flexible aggregate with an electron microscope. Formation of an Mg-polymcrwas once believed to be specific for plasmodiumactin, but it was shownsubsequently that this type of polymer was formed only when the actin preparation contained a cofactor similar to the muscle fl-actinin (90). This protein factor was isolated from plas- modiumand called "fl-actinin-like protein" (110) or plasmodiumactinin (41). Recently, Hasegawaet al (33) have further purified this protein factor and found that it is a 1 : 1 complex of actin and a new protein termed "fragmin" (scc later). This protein is shownto have a regulatory function Annual Reviews www.annualreviews.org/aronline

212 KAMIYA

in the formationof F-actin filaments in a Ca2+-sensitivemanner. Hence, there is the possibility that the so-called "Mg-polymer"of actin wasformed by a trace amountof Ca2+. Detailed properties and polymerizability of plasmodiumactin are discussed by Nachmias(116), Hatanoet al (37) Hinssen(55) in a recent bookedited by Hatanoet al (36).

Tension Production of Reconstitute d Actomyosin Threads from Physarum Physarumactomyosin dissolved in a solution of high ionic strength precipi- tates in the formof threadif spurtedfrom an injection needleinto a solution of low ionic strength. Becket al (15) showedthat the thread consists of three-dimensionalnetwork of filaments, has ATPaseactivity, and contracts conspicuously on addition of ATPjust like muscle actomyosinthread. D’Haese& Hinssen (25) comparedthread modelsmade of natural, recom- bined, and hybridized actomyosinfrom Physarumand rabbit skeletal muscle. Recently, Matsumuraet al (112) were able to reconstitute actomyosinthread from Physarumwith an orderly longitudinal orienta- tion, and to measurethe tension it developedunder controlled experimental conditions. Toinsure orderly longtudinalorientation of actomyosin,which is essential for the thread to generatetension effectively, they developeda special spinning technique applicable to aetomyosin. Thread segments of actomyosin(molar ratio 1:1) thus obtained produced little tension below 1 #MATP, whereas maximumtension (10 em-2) was reached at 10 /.tM ATP. The half-maximumtension was observed at 2-3 #MATP. Above20 #MATP, the thread segment tended to break. Withoutan ATP-regeneratingsystem, the sensitivity of tension developmentto ATPconcentration was lower by one order. Full tension at 20 /zM ATPdecreases as the ATPconcentration is decreased stepwise to 1 /.tM. Whenthe ATPconcentration was increased

by STEWARD OBSERVATORY on 04/23/06. For personal use only. from1 to 10 btM,the decreasedtension rose again to almostthe samelevel as that originally developed.In short, isometric tension generationcan be regulated by the mieromolarconcentration of ATP.

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org The aetomyosin thread from Physarumdiffers from that of skeletal musclein several ways(24, 112). 1. The Physarurnactomyosin thread is muchmore flexible; 2. the concentrations sufficient to produce maximum and half-maximumtension are lower than those reported for muscle ac- tomyosinthreads, for whichthese values are 50 and 8 raM,respectively; 3. tension increase in Physarumactomyosin thread is slower than that in muscleaetomyosin thread, wherethe final level of tension is reachedwithin 2 rain. Thesefunctional differences can probablybe ascribed to the differ- enee in properties betweenPhysarum and musclemyosin, such as the high solubility of ~Physarurnmyosin at low ionic strength, a propertynot found in muscle myosin. Annual Reviews www.annualreviews.org/aronline

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Since their preparation of synthetic actomyosinwas highly purified, the thread used by Matsumuraet al (I 12) showedno 2+ sensitivity in tension generation at micromolar levels. Under their experimental conditions, in which no regulatory proteins were present, there was no sign of oscillation in tension production at constant ATPlevels. Whetheroscillation in tension production is possible in a reconstituted Physarumactomyosin thread in the presence of appropriate regulatory proteins, but without a membrane system, is still an open question. It should be noted that in the demembranated system of Physarum plasmodium studied by Kuroda (103, 104), cytoplasmic movementoccurred actively, but there was no longer any back and forth movementas is observable in the normal plasmodiumhaving the plasma membrane.

Regulation of Movement Various regulatory functions can be seen in the force output of the slime mold, as stated in the foregoing pages, such as activation through stretching or loading or phase coordination of tension force production. The cause of rhythmic tension force production mayin itself be inseparable from the mechanismregulating interaction between actin and myosin. Thoughnoth- ing definite is knownat present about the regulation mechanismof move- mcnt in the slime mold, we should like to consider in the following sections some possible roles of Ca2+ and ATP.

THEROLE OF Caz+ Isolation of Ca2+-sensitive actomyosin complexes . from Physarum(92, 117) suggests that the actin-myosin interaction is controlled by fluctuation in the concentration of intraceLlular free Ca2+ (174). The control of motility in Physarumby calcium can be demonstrated in various ways. It was shown that in the myxomyceteplasmodium there is a calcium storage system analogous to the sarcoplasmic reticulum (91, 94). Calcium-sequestering vacuoles were identified both by histochemical meth- by STEWARD OBSERVATORY on 04/23/06. For personal use only. ods and by energy-dispersive X-ray analysis (17, 18, 29, 106, 107). 2+ is taken up by the vesicles only in the presence of Mg2+-ATP(91, 94). It possible that there is a shift of calcium betweenthe cytoplasmic and vacuo- Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org lar compartments during the contraction-relaxation cycle. Teplov et al (149) were successful in detecting oscillations of the free calcium level the myxomyceteplasmodium by injecting murexidein it. Oscillations in the Caz+ level within the period of 1.5-2.0 rain were demonstrated by micro- spectro-fluorometry of injected murexide, although the phase relation between the Ca2+ level and motility was unknown. Ridgway& Durham(137) microinjected the calcium-specific photoprotein aequorin and found an oscillation in luminescence related to that in electric potential change. Ca~+ regulation is shownalso in catfein-derived microplasmodialdrops (34, 113), in microinjected strands (152), and in a demembranatedsystem (103). Annual Reviews www.annualreviews.org/aronline

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In a recent attempt to monitorthe calciumosdllation in the slime mold and to relate it directly to tension production, Kamiyaet al (89) and Yoshimotoet al (178) treated a segmentof plasmodialstrand with calcium ionophore A 23178. They simultaneously measuredtension development and, by meansof aequorinluminescence, the calciumeiflux into the ambient solution. It was revealed that the amountof calcium comingout of the plasmodial strand pulsates with exactly the same period as that of the tension production, and that the phase of maximaltension production corresponds to the phase of minimalluminescence. With amphoterieinB, a channel-formingquasi-ionophore (135), the result wasessentially the same. Regularrhythmic changes in both tension and luminescencepersisted for hours. In the absenceofionophore, no periodic Ca2+ efltux wasdetected fromthe strand developingtension rhythmically(79, 109): Asis generally the case with the plasma membrane,the surface membraneof the plas- modinmalso depolarizes whenit is stretched. Onstretching the strand by 10%or so, the tension levd of the strand and amplitudeof tension oscillation were immediatelyincreased. But Ca2+ efl]ux was not affected. This result maymean that electric potential differenceplays little part, if any, in controlling the etttux of Ca2+. Probablythe membranealready has depolarizedin the aboveconditions. If weinterpret the rhythmicaletitux of Caz+ in the presenceof the ionophoresas reflecting correspondingfluc- tuations of free Ca2+ level withinthe plasmodium,this phaserelation is just the opposite of what is expectedfrom the data so far presented. In this connection,it is interesting to note that "fragrnin," a newCa2+-sensitive regulatory factor in the formationof actin filaments, wasrecently discovered by Hasegawaet al (33). Themain function of this protein is to fragment aetin polymersinto short pieces in the presenceof a concentrationof free Ca:+ higher than 10-6M. Whether or not fragmin plays a part in the regulation by Ca2+ of cyclic tension output is unknown. by STEWARD OBSERVATORY on 04/23/06. For personal use only. THEROLE OF ATPThe ATPconcentration of plasmodia as a total mass was~stimated to be 0.4 X 10~ M(44). Accordingto the injection experiments in the plasmodial strand performedby Ueda& G6tz yon Olenhusen Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org (152), the optimal concentration for tension developmentwas found to around 0.2 X 10-4 M. Accordingto the recent ATPassay by Yoshimotoet al (unpublished),using luminescenceof lucfferin-luciferase, a considerable part of the ATPin Physarumplasmodia is compartmentalized.The free ATPconcentration in a carefully prepared homogcnatcof Physarumplas- modiumwas low, but ff the samehomogenate was heated in boiling water, the intensity of luminescencewas suddcrdy increased by nearly two orders of magnitude.This result is interpreted as showingthat compartmentalized ATPwas released by heat. In other words, free ATPavailable for mechan- Annual Reviews www.annualreviews.org/aronline

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ical work in vivo must be at a muchlower level than the total average. The fact that the optimal ATPconcentration for tension development by a reconstituted actomyosin thread is as low as 10-20 pM(112) is also conformity with this notion. There is someevidence to showthat the level of free ATPoscillates with the same period as tension changes. As they had done for Ca2+, Kamiya et al (89) and Yoshimotoet al (178) tried to make the surface membrane of the plasmodial strand leaky for ATP. The combinedaction of caffeine and arsenate was found to be effective. Simultanously with tension measurement, they measured the amount of ATPdiffusing out of the strand by the luminescenceof a luciferin-lucifcrasesystem. The plasmodial strand could notpcrsist long in thc caffcinc-arscnatc solution, butit couldexhibit at Icast severalregular waves of tensionproduction accompanied by simultaneous oscillationin lumincsccncc forI0-20 rain before it undcrwcntirrcvcrsiblc damage.In thiscase, the tension maxima colncldcd well with the lumines- cencemaxima, and tcnsionminima with thc lumincsccnccminima. Under normalconditions, thcrc was no dctcctablcleakage of ATP.Oscillation in lightemission caused by ATPreleased from the strandis provisionally intcrprctcdas reflecting changes in thelevel of frccATP within the plasmodium.This interpretation is supported by theresults obtained recently by T. Sakai(unpublished), who measuredATP levelsof the plasmodial strandsin contractionand relaxation phases separately with a luciferin- lucifcrascsystem; hc showedthat the ATP level was statistically signifi- cantlyhigher in thephase of maximaltension than in thephase of minimal tension. Theshift in thephases of oscillationsin Ca 2+ andATP efliuxcs by just 180° is an interestingproblem. Although we do notknow the cause of this phaserelationship, wc arc reminded that the calcium-activated ATPpyro- phosphohydrolasc(APPH) characterized by Kawamura& Nagano(96) existsin the P~tysarumplasmodium. Perhaps if APPHis activatedwith an by STEWARD OBSERVATORY on 04/23/06. For personal use only. increaseof Ca2+concentration, theconcentration of ATP will be decreased; inversely,if APPHis suppressedwith a decreasein Ca2+ level,the ATP levelwill bc increased.This is mcrclyspeculation on the inverse relation Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org betweenCa 2+ and ATP concentrations. In analogyto musclecontraction, it is generallybclicvcd that micromolar Ca2+ stimulatesrather than inhibits streaming. Thc weightof cvidcncc so farknown is favorableto thisview, but wc stillcannot rule out the possibilitythat Ca 2+can act as a suppressorof movement by wayof control- lingthe ATP level. As a matterof fact,micromolar Ca2+ is inhibitoryto streamingin thecase of Nite/[a,as we shallscc later. Although there arc stillsome ambiguities about the roles of Ca2+ andthe possibility of ATP limitingthe rate of forceoutput, it seemsreasonably safe to saythat in- Annual Reviews www.annualreviews.org/aronline

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tracellular concentrationsof Ca2+ and ATPavailable to actomyosinchange in vivo with the sameperiod as tension development. Structural Basis of Motility Dynamicactivities of the slime moldare closely connectedwith morphological changeson the part of microtilamentsin the cytoplasm.It wasshown for the first time by Wohlfarth-Bottermann(168-170) that in the myxomyceteplasmodium there are bundles of microfilaments with a diameter of abont 7 nm. Theyare foundin the ectoplasmand terminateat the surface of invaginated membrane.There is substantial evidenceto showthat most of these bundles are composedof plasmodialactomyosin and that they are the morphologicalentities responsiblefor the contractility in this organism (13, 14, 122).

CONTRACTION-RELAXATION CYCLE AND ACTIN TRANSFORMA- TIONS Combiningtension monitoring of the plasmodial strand with electron microscopy,various workershave shownthat the microfilamentsand their assembly undergo remarkable morphologicalchanges in each contraction-relaxationcycle (30, 124, 125, 172). In the shorteningphase of the strand under isotonic contractions, the membrane-boundmierofilaments with a diameterof 6-7 nmare nearly straight and arrangedparallel to one another to form large compact bundles whoseadjacent filaments are bridged with cross linkages (124, 125). Amongthese microfilaments, thicker filaments whichare thought to represent myosinbundles are sporad- ically scattered (14, 124, 125). Whenthe strand approachesthe phase maximalcontraction underisotonic conditions, mostof the microfilaments becomekinky and form networks(124, 125). In the elongation phase, new loose bundlesof microfilamentsdevelop from the network. Parallelization of the loose bundlesis completedby the time the strand reaches its maximal

by STEWARD OBSERVATORY on 04/23/06. For personal use only. elongation. Theybecome compact again in the contracting phase, until they lose their parallel order and becomeentangled to formthe networkas the maximalcontraction is approached. Recent microinjection experiments

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org using phalloidin conductedby G/Stz von Olenhusen& Wohlfarth-Botter- mann(32) also showthe involvementof actin transformationsin the contraction-relaxation cycle of tension development. Wohlfarth-Bottermannand his group are of the opinion that actin may undergoG-F transformation in each contraction-relaxation cycle (173). Since endoplasm-ectoplasmconversion is constantly occurring in the mi- grating plasmodium,especially in its front and rear regions, and the plas- modiumcontains a considerable amountof G-actin, it is reasonable to supposethat G-Ftransformation occurs locally. Whethercyclic G-Ftrans- formation is a predominantfeature occurring with cyclic tension produe- Annual Reviews www.annualreviews.org/aronline

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tion in a system like the plasmodial strand segment, however, is unknown. Probably plasmodiumactinin (41), fragmin (33), or someother regulatory proteins (S. Ogihara and Y. Tonomura,unpublished) play an important role in modifying the state of actin polymers in each cycle of tension development.

FIBRILLOGENESISIn his early observations of the strand hung in the air, Wohlfarth-Bottermann (170) noticed that there are a larger number fibrils (microfilament bundles) in the ectoplasmic gel tube whenthe endoplasm flows upward against the gravity than when it flows downward assisted by the gravity. Thus there seemedto be a direct relationship between the amount of motive force needed and the number of fibrils. This notion put forth by Wohlfarth-Bottermann (170) has been supported further experiments (30, 76). If we apply extra load to the strand, more fibrils emerge. This is a sort of morphologically detectable regulation in response to the load applied. A favorable material in whichto study the de novo formation of the fibrils is a naked drop of endoplasmformed after puncturing the strand (or vein) (61). Soon after the protrusion of the endoplasm, the drop has a low viscosity and no bundles of microfilaments. A considerable amountof actin is thought by someworkers to be in a nonfilamentous state at first; but polymerization of actin gradually proceeds to form bundles of F-actin. On the question of whether F-actin is present in an endoplasmicdrop from the beginning, opinions are not unanimous (122). At any rate, new bundles F-actin are developed within 10 rain from the "pure" endoplasmic drop to form the ectoplasm. An isolated drop originating from the endoplasmin the mother plasmodium has now become an independent plasmodium having the normal endoplasm-ectoplasmratio and exhibits shuttle streaming and migration. This fibdllogenesis is an interesting exampleof regulation on the

by STEWARD OBSERVATORY on 04/23/06. For personal use only. organizational level of cytoplasm.

BIREFRINGENCECyclic changes in filament assembly are demonstrated also by changes in birefringence detectable in the living plasmodiumwith Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org a highly sensitive polarizing microscope. In a thin fan-like expanse of the spread plasmodium,appearance and disappearance of the birefringent fibers are repeatedly observed in the same loci in the cytoplasm accompaniedby back and forth streaming. These birefringent fibrils could be stained with rhodamin¢-heavy meromyosin (R-HMM).Further, 0.6 KI readily made the birefdngent fibrils disappear (127). Thus it was confirmed that these birefdngent fibers represent bundles of F-actin. In the contracting phase of the expanse of plasma, in which streaming takes place away from the front (backwardstreaming), stronger birefringence appears than in the relaxing Annual Reviews www.annualreviews.org/aronline

218 KAMIYA

phase (forward streaming) in which the streaming takes place toward the advancing front. In the latter phase, birefringenee fades away or nearly disappears, except that some thicker fibers forming knots or compact regions remain in situ (76). This behavior of the fibrous structure on the optical microscope level agrees well with the observation by electron microscopy. Disappearance or weakeningof birefringence is interpreted to reflect deparallelization of the micro filaments or their depolymerization. Localization of the contractile zone in glyeerinated specimen (86) also coincides with the distribution and population of the birefringent fibers. Hinssen & D’Haese(58) obtained birefringent synthetic fibrils from Physa- rum actomyosinwhich resemble in size and fine structure the fibrils in vivo.

Important points so far knownabout cytoplasmic streaming in the myxomycete plasmodium may be summarized as follows:

1. The streaming is caused by a local difference in internal pressure of the plasmodium.This pressure difference, or the motive force responsible for the streaming, is measurable by the double-chamber method. 2. The internal pressure is brought about by the active contraction of the ectoplasmic gel. 3. Contractile force of the ectoplasmic gel can be measuredas a unidirectional force in a segment of plasmodial strand both under isometric and isotonic conditions. 4. Tension oscillation is augmentedconspicuously by stretching or by loading. 5. The structural basis of contraction is the membrane-boundfibrils in the ectoplasm. 6. The fibrils consist mainly of bundles of F-actin and of smaller amounts

by STEWARD OBSERVATORY on 04/23/06. For personal use only. of myosin and regulatory proteins. 7. Tension2+. development is regulated by Ca 8. Actin filaments undergo cyclic changes in their aggregation pattern

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org and/or G-F transformation in each contraction-relaxation cycle. 9. Physarumactin is strikingly similar to rabbit striated muscleactin in its properties, including amino acid composition and sequence. 10. Physaruramyosin is also similar to rabbit striated muscle myosinin its molecular morphology and ATPase activity, but Physarura myosin is more soluble at low salt concentrations than muscle myosin. Pt~ysarum myosin self-assembles to form bipolar thick filaments which are less stable than those of rabbit skeletal muscle. 11. Phyaaruraactomyosin undergoes reversible superprecipitafion on addition of Mg-ATP.This characteristic is not shared with muscle actomyosin. Annual Reviews www.annualreviews.org/aronline

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12. The actomyosin thread derived from Physarumcontracts on addition of Mg2+-ATP.It produces tension as strong as 10 g cm-2, which is comparable to that of a muscle actomyosin thread at the same protein concentration. 13. The aggregation pattern of actin filaments and concentrations of free calcium and free ATPoscillate with the same period as cyclic tension generation. In addition, electric potential difference (79), pHimmedi- ately outside the surface membraneNakamura et al, unpublished), and heat production (12) are also knownto change with the same period as shuttle streaming. Howthese physiological parameters are causally related, and whatthe origin of oscillation is, still are unknown(16, 172).

ROTATIONAL STREAMING IN CHARACEAN CELLS General The other type of streaming which has been studied extensively recently is rotational streamingin giant eharaceancells. It is in this groupof cells that Corti discovered cytoplasmic streaming for the first time in 1774. This type of streaming is quite different from that in the slime mold, in that the cytoplasmflows along the cell wall in the form of a rotating belt, with no beginning or end to the flow. Since the cells of Characeaeare large and the rate and configuration of the streaming in them are nearly constant, they lend themselves favorably to physiological and biophysical experiments. The Site of Motive Force Generation Each of the two streams going in opposite directions on the opposite sides of the cylindrical cell has the samerate over its entire width and depth to within close proximity of the so-called indifferent lines. There the two opposed streams adjoin and the endoplasm is stationary (73). In other words, the whole bulk of endoplasmmoves as a unit, as though it slides by STEWARD OBSERVATORY on 04/23/06. For personal use only. along the inner surface of the stationary cortical gel layer. Only the narrow boundary layer between the endoplasmand cortex and the cell sap between the two opposing streams are sheared. Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org In the endoplasm-filled cell fragment, whichcan be obtained artificially by gentle eentrifugation and subsequent ligation, the endoplasmitself is sheared to showa sigmoidvelocity profile similar to that of the cell sap. The highest velocity is found in this case at the region in direct proximityto the stationary cortex. This velocity profile coincides exactly with what is expected whenan active shearing force (parallel-shifting force) is produced opposite directions at the boundary regions on the opposite halves of the cell and the rest of the endoplasmis carried along passively by this periph- eral force (81). That the endoplasmin the endoplasm-filled cell, or the cell sap in the normal internodal cell, shows the velocity profile of a sigmoid Annual Reviews www.annualreviews.org/aronline

220 KAMIYA

form is simply a function of the cell’s cylindrical geometry. As a matter of fact, whena part of the cylindrical cell is compressedand flattened between two parallel walls to such an extent that the two opposing flows come in contract and fuse, the velocity profile is no longer sigmoidbut straight (85). Weshall discuss this problem again later. The simple and unique conclusion derived from these experimental facts is that the motiveforce driving the endoplasmin the Nitella cell is an active sheafing force producedonly at the interface betweenthe stationary cortex and the endoplasm (71, 81). Naturally this model does not specify the nature of the force or the mechanismof its production, but stresses the importance of interaction between cortex and endoplasmfor the production of the motive force. Endoplasmaloae is not capable of streaming, as wiLl be described in a later section. It should be noted, however, that the above model is not the only current view regarding the site of the motive force. N. S. Allen (5, 6, 8) observed the bending waves of the endoplasmic filaments traveling in the direction of streaming and the particle saltation along these filaments. She believes that both play a substantial part in producing the bulk flow, in addition to the force produced at the ¢ortex-endoplasm boundary. Thus she raised an objection to the above model of active shearing. The phenomenonwhich N. S. Allen described appears in itself fascinating and intriguing. The correct- ness of a theory on the site of the motive force, however, must be based upon a consistent explanation of the intracellular velocity distributions under defined conditions. If any force participated substantially in driving the endoplasmat loci other than its outermost layer, the flow profiles wouldbe distorted from what we have actually observed.

Subcortical Filaments, Their Identification as Actin, and Their Indispensability for Streaming

by STEWARD OBSERVATORY on 04/23/06. For personal use only. Ten years after Kamiya& Kuroda (81) concluded that the motive force produced in the boundary between the stationary cortex and outer edge of the endoplasm in the form of active shearing, fibrillar structures were

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org discovered at the exact site where the motive force was predicted to be generated. This discovery was madealmost simultaneously by light microscopy (66, 68) and electron microscopy(123). The fibrils are attached to inner surface of files of chloroplasts whichare anchored in the cortex. They run parallel to the direction of streaming. Later the subcortical fibrils were visualized also with scanning electron microscopy(9, 98). Each of the fibrils is composedof 50-I00 microfilaments with a diameter of 5-6 nm (123, 132). These microfilaments were identified as F-actin by Palevitz et al (130), Williamson (163), Palevitz & Hepler (131), and Palevitz (129) through formation of an arrowhead structure with heavy meromyosin (HMM) Annual Reviews www.annualreviews.org/aronline

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subfragment I of HMM.Further, it was shown by Kersey ¢t al (97) that all these arrowheadspoint upstream. Thesubcortical fibrils were also identified as aetin by an immunofluoreseeneetechnique (37, 167). Aninteresting approach to studying functional aspects of the subeortieal fibrils is to destroy or dislodge the fibrils locally by such meansas mi- erobeamirradiation (67), centrifugation (69), or instantaneous aeeeleration with a mechanical impact (84). The endoplasmic streaming is either stopped or rendered passive at the site where the filaments disappear. The streaming resumes only after the filaments have regenerated. The new streaming always occurs alongside the newly regenerated filaments and not dsewhere (67-69). All these facts showthat subcortical filaments are essential for streaming. Further evidence in support of this view is to be had from differential treatment of an internodal cell with cytochalasin B (121) (see later). Nitella Myosin and its Localization in the Cell Voroby’ eva & Poglazov (158) showeda slight viscosity drop upon addition of ATP in a myosin B-like protein extracted from Nitella. Kato & Tonomura(95) demonstrated the existence of myosin B-like protein Nitella more definitely, and characterized its biochemical properties. At higher ionic strength, ATPaseactivity of the protein complexwas enhanced by EDTAor Ca2+ and inhibited by Mg2+. At low ionic strength~ superprecipitation oceurred with the addition of ATP.Myosin was further purified by Kato and Tonomura from Nitella myosin B. The heavy chain of Nitella myosinhas a molecular weight slightly higher than that of skeletal muscle myosin. Recently myosins were also extracted from the higher plants Egeria (Elodea) (128) and Lycopersicon (156), All these myosins possessed aetin-stimulated ATPaseactivity and the ability to form bipolar aggregates. An important problem in the mechanismof cytoplasmic streaming is the by STEWARD OBSERVATORY on 04/23/06. For personal use only. intraeellular localization of myosinand the modeof its action. Aneffective physiological approach is to treat the streaming endoplasmand the stationary cortex of the living internodal cell of Nitella separately with a reagent Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org whoseaction on actin and myosin is clearly different, and to see howthe streaming is affected. A method combining the double-chamber technique with a pair of reeiproeal eentrifugations madesuch differential treatment possible (22, 77). Whenan internodal cell is centrifuged gently, the endoplasm collects at the centrifugal end of the cell while the cortex, ineluding ehloroplasts and subortieal fibrils in the centripetal half of the cell, remains in situ. Then either the centrifugal half or centripetal half of the cell is treated with an appropriate reagent. Finally, the treated endoplasmis trans- located by a second centrifugation in the reverse direction to the other end Annual Reviews www.annualreviews.org/aronline

222 KAMIYA

of the cell to bring it in contact with the untreated cortex; by the same process, untreated endoplasmis broughtinto contact with treated cortex. It is knownthat N-ethylmaleimide(NEM) inhibits F-actin-aetivated ATPaseof myosin,but this reagent has little effect on polymerizationor depolymerizationof actin. Whenthe cell cortex wastreated with NEMand the endoplasmwas left untreated, streamingoccurred normally. Whenonly endoplasmwas treated with the samereagent, no streamingtook place (22, 77). If the cell wastreated differentiallywith heat (47.5°Cfor 2 rain) instead of NEM,the result wasessentially the sameas in the case of NEM(J. C. W. Chenand N. Kamiya,unpublished). This is because myosinis readily denaturedby heat while actin is heat-stable. Whenwe used cytochalasin B (CB), whoseinhibitory effect on the cytoplasmicstreaming in plant cells well known(19, 21, 164), the situation wasexactly the opposite. Cytoplas- mic streaming was stopped only whencortex was treated with CB(121); whenendoplasm alone wastreated with this reagent, streaming continued. Theimplication of these results is that the componentwhose function is readily abolished by NEMand heat must be present in the endoplasm,not in the cortex. It is not incorporated with the subcortical microfilament bundles. This componentis presumably myosin. The componentwhose function is impairedby CBmust reside in the cortex, not in the endoplasm. This result shows,as already mentioned,that actin bundlesanchored on the cortex are indispensablefor streaming. Motility of Fibrils and Organdies in Isolated Cytoplasmic Droplets A nakedendoplasmic droplet squeezedout of the cell into a solution with an ionic compositionsimilar to the natural cell sap forms a newmembrane on its surface and can survive for morethan 24 hours, sometimeseven for several days.

by STEWARD OBSERVATORY on 04/23/06. For personal use only. In the isolated drop of endoplasmcontaining no ectoplasm,there ran be seen what appears as Brownianmovement and agitation (saltation) minute particles; but, as expected, there is no longer any sign of mass streaming of the endoplasmitsdf. This is a further confirmation of our Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org previousconclusions (71, 8 I), that rotational cytoplasmicstreaming within the cell takes place only whenthe endoplasmcomes in contact with the cortical gel layer and that the bulk of the endoplasmis passively carried along by the force producedat its outer surface.

ROTATIONOF CHLOROPLASTSA startling phenomenon in the endo- plasmiedroplet is the independentand rapid rotation of ehioroplasts. This phenomenonwas observed by several authors long ago and described in detail by Jarosch for squeezedout cytoplasmicdroplets (63-65) and Annual Reviews www.annualreviews.org/aronline

CYTOPLASMICSTREAMING 223

Kuroda(102) for isolated endoplasmicdroplets containingno cortical gel. Theswifter chloroplasts makeone rotation in less than one second. When wecarefully observethis rotation by meansof an appropriateoptical system, we find that in the layer immediatelyoutside the rotating bodythere occurs a streaming in the direction opposite to that of rotation. When rotation is prevented by holding the chloroplast with a microneedle, a counter streamlet becomesmanifest along the surface of the chloroplast (102). Whenthe chloroplast is allowedto rotate freely again, the counter- streamingbecomes less evident. Theseobservations are further proof for the existenceof an active shearingforce betweensol (endoplasm)and gel (chlo- roplas0 phase. Therotation of chloroplasts is attributable to the attach- mentto their surface of actin filaments, whichare the sameas subcortical fibrils. Usingisolated droplets of endoplasm,Hayama & Tazawa(50) investigated the effect of Ca2+ and other cations on the rotation of chloroplasts by injecting themiontophoretically. Uponmicroinjection of Ca2+, chloroplast rotation stoppedimmediately but recoveredwith time, suggestingthat a Ca2+~sequesteringsystem is present in the cytoplasm. These authors estimatedthe Ca2+ concentrationsnecessary for halting the rotation to be > 10-4M.Sr 2+ had the same effect as Ca2+. Mn2+ and Cd2+ also slowed downthe rotation, but the effect wasgradual and the reversibility waspoor. K+ and Mg2+ had no effect. These results show that Ca2+ acts as an inhibitor rather than activator of the movementin Nitella. Perfusionexperi- ments(see later) also showthe samething.

MOTILEFIBRILS When we observe a cytoplasmic droplet that has been mechanicallysqueezed out of a cell of Nitella or Charaunder dark field illumination with high magnification,we find in it a baffling repertory of movementson the part of extremely fine fibrils which maytake the form

by STEWARD OBSERVATORY on 04/23/06. For personal use only. of long filaments or closed circular or polygonalloops. Theremarkable behaviorof the motilefibrils has beendescribed in detail by Jarosch(63-65) and Kuroda(102).

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org Importantcharacteristics of these fibrils are their self-motility andtheir capacity to formpolygons. Minute particules (spherosomes)slide (saltate) alongside these fibrils. Their movementsare again producedby a mechanism of countershifting relative to the immediatelyadjacent milieu, as pointed out earlier by Jarosch (63). Kamitsubo(66) confirmedthat a freely rotating polygonalloop emergesfrom the linear stationary fibrils in vivo through"folding off." His observation verifies the viewthat the motile fibrils and rotating loops or polygonsin the droplets are the samein nature as the stationary subcorticalfibrils. Thesemotile filaments are identified, through arrowhead formation with HMM(54), as being composedof Annual Reviews www.annualreviews.org/aronline

224 KAMIYA

actin. The endoplasmic filaments observed by N. S. Allen (7) in vivo seem to be also of this kind, branching off from the cortical filament bundles.

Demembranated Model Systems The outstanding merit of using a demembranated cytoplasm is that the chemicals applied presumably diffuse readily into the cytoplasm without being checked by the surface plasma membraneor by the tonoplast. REMOVALOF TONOPLASTElY VACUOLARPER-FUSION Taking ad- vantage of the technique of vacuolar peffusion developed by Tazawa044), experiments were done recently to remove the vacuolar membraneby using media containing ethyleneglycol-bis-(~-aminoethylether)N,N’-tctraacetate (EGTA)(146, 164). This is a sort of cell model, like the demembranatcd cytoplasmic droplets to be mentioned later. These experiments show that endoplasmremaining in situ shows a full rate of active flow if the perfusing solution fulfils certain requirements. The presence of ATPand Mg2+ is indispensable. Williamson (165) showedthat generation of the motive force is associated with the subcortical fibrils. On addition of ATP,endoplasmic organelles and particles adhering to the fibrils start movingalong the fibrils at speeds up to 50 ~ms -1, but are progressively freed from contact, leaving the fibrils denuded. It was shownthat streaming requires a millimolar level of Mg2+ and free Ca2+ at 10-7 Mor less. Higher concentrations of Ca2+ were inhibitory (49, 146). Hayamaet al (49) concluded that instantaneous cessation of the streaming upon membraneexcitation is caused by a transient increase in the Ca2+ concentration in the cytoplasm. Nitella cytoplasm contains about 0.5 mMATP (38). Shimmen(140) showed, using tonoplast-free cells, that the ATPconcentration for half saturation of streaming velocity was 0.08 mMin Nitella axillaris and 0.06 mMin Chara eorallina. Mg2+ is essential at a concentration equal to or higher than that of ATP,whereas Ca2+ concentrations in excess of 10-7 M by STEWARD OBSERVATORY on 04/23/06. For personal use only. are inhibitory to streaming. Inhibition of streaming by depletion of ATPis completely reversible, but that caused by the depletion of free Mg2+ is irreversible. This observation suggests that Mg2+ mayhave a role in main- Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org taining somestructures(s) necessary for streaming, besides acting as eofae- tor in the ATPasereaction (140).

DEMEMBRANATEDCYTOPLASMIC DROPLETS Taylor et al (142, 143) conducted a series of important observations on amoeboidmovement, developing improved techniques of demembranation. In the case of Nitella droplets, it is also possible to remove the surface membranewith a tip of a glass needle in a solution with low Ca2+ concentration [80 mMKNO3, 2 mM NaCI, 1 mM Mg(NO3)2, 1 mMCa(NO3)2, 30 mM EGTA, ATP, 2 mMdithiothreithol (DTT), 160 mMsorbitol, 3% Ficoll, 5 Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 225

Pipes buffer (pH 7.0)] (105). As the cytoplasm gradually dispersed into surrounding solution, the earlier dear demarcation of the droplet disap- peared, but chloroplasts continued to rotate almost as actively as they did before the surface membranewas removed. Whena chloroplast came out spontaneously from the cytoplasm-dense region into the cytoplasm-sparse area beyond the original boundary, the rotation becameslow and sporadie. The rotation of chloroplasts that emerged from the core area stopped completely on addition of 1 mMN-ethylmaleimide (NEM)to the above solution. Chloroplast rotation did not recover even though free NEMwas removed with an excess amount of DTT. This was probably because NEM inhibited F-actin-activated ATPase of myosin, forming a covalent bond with SH-1 of myosin. Kuroda & Kamiya(105), however, observed that the rotation of chloroplasts resumed, even though the rate of rotation was slow and somewhat sporadic, if rabbit-heavy meromyosin (HMM)was added the solution in the presence of Mg2+-ATP.It took 1 min or longer for a chloroplast to complete one revolution. The rotation of chloroplasts lasted only 10 rain or so, but this fact implies that chloroplast rotation in vitro, and hence cytoplasmic streaming in vivo, results from the interaction between F-actin attached to the chloroplast and myosin in the presence of Mg2+-ATP.It also shows that Nitella myosincan be functionally replaced, to some extent at least, with muscle HMM(105).

ACTIVE MOVEMENTIN VITRO OF CYTOPLASMICFIBRILS Another interesting observation on the movementof chloroplasts and cytoplasmic fibrils were made recently by Higashi-Fujime (54). In the demembranated system in an activating medium (1.5 mMATP, 2 mMMgSO4, 0.2 sucrose, 4 mMEGTA, 0.1 mMCaCl2, 60 mMKCI, 10 mMimidazole buffer pH 7.0), chloroplast chains linked together by cytoplasmic fibrils movedaround at the rate of about 10 btm/see for 5-10 min. Dark field

by STEWARD OBSERVATORY on 04/23/06. For personal use only. optics showedthat free fibrils curved and turned but never reversed. Travel- ing fibrils occasionally were converted into a rotating ring. All of the rotating rings, movingfibrils, and fibrils connecting chloroplasts in chains were shownto be composedof bundles of F-actin. Each bundle of F-actin Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org had the same polarity as revealed by decoration with rabbit muscle HMM. Addition of HMMfrom rabbit skeletal muscle did not accelerate their swimmingvelocity (S. Higashl-Fujime, personal communications).

Measurement of the Motive Force The rate of flow, which is often used as the criterion for the activity of cytoplasmicstreaming, is a function of two variables: motive force responsible for the streaming, and the viscosity of the cytoplasm. Therefore, if the rate of flow is changed under a certain experimental condition, we do not Annual Reviews www.annualreviews.org/aronline

226 KAMIYA

knowto what extent it is due to the change in the motive force and to what extent to the change in viscosity. Since there is no method for measuring exactly the viscosity of cytoplasm in vivo, it is extremely important for understanding the dynamics of cytoplasmic streaming to measure the motive force. So far three methods have been developed for measuring the motive force responsible for rotational streaming in Nitella.

CENTRIFLIGATIONMETHOD (82) When an internodal cell is centrifuged along its longitudinal axis, the streaming toward the centrifugal end is accelerated while the streaming toward the centripetal end is re- tarded. Throughcentrifuge microscope observation it is possible to deter- mine the centrifugal acceleration at which the streaming endoplasmtoward the centripetal end is brought just to a standstill. Measuringthis balance- acceleration and the thickness of the layer of the streaming endoplasm, and knowing the difference in density between the endoplasmand cell sap, we can calculate the motive force, i. e. the sliding force at the endoplasm-cortex boundary per unit area. It is found usually to be between 1.0-2.0 dyn cm-~ (82). This is the method which made possible the measurementof the motive force of rotational cytoplasmic streaming for the first time.

PER.FrUSIONMETHOD (145) When both ends of an adult Nitella internode are cut off in an isotonic balanced solution and a certain pressure difference is established betweenthe two openings of the cell, the vacuole can be perfused with that solution. This flow of fluid exerts a shear force upon the vacuolar membrane(tonoplast) and accelerates the endoplasmic streaming in the direction of perfusion on one side of the cell, but retards the streaming on the other side. The greater the speed of perfusion, the slower is the rate of streaming against it. At a certain perfusion rate, the endoplasmic streaming against it is brought to a standstill. The sheafing

by STEWARD OBSERVATORY on 04/23/06. For personal use only. force producedat the tonoplast under this state must be nearly equal to the motive force produced at the endoplasm-ectoplasm interface, because the thickness of the endoplasmiclayer of the adult Nitella internode is small

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org as comparedwith the radius of the vacuole. The motive force thus measured by Tazawawas found mostly in the range of 1.4--2.0 dyn cm-2. This range is in excellent agreement with the values obtained by the centrifugation methodin the same material (82). By using a theoretical modal and assump- tions regarding the form of stress/rate of strain curve (85), Donaldson(27) determined possible velocity distributions for the streaming eytopiasm in the normal cell and in the cell whose vacuole is perfused. Based on a mathematical analysis, he estimated the motive force to be 3.6 dyn em-2 and the thickness of the motive force field (high shear zone) betweenthe endoplasm and cortical gel layer to be 0,1 /zm. Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 227

With the perfusion method, it was shownthat the motive force is almost independent of temperature within the range of 10°-30°C, while flow velocity increases conspicuously with the rise in temperature (145). This result showsthat the change in the rate of flow as the temperature varies, is, at least in the case of characeancells, mainlythe result of a changein viscosity of the fluid in the high shear zone at the endoplasm-ectoplasmboundary and not to the change in the motive force. Increase in viscosity of the cytoplasm, however, is not always the cause of the slowing downor cessation of flow. For instance, the slackening of the streaming through application of an SH reagent, p-chloromercuribenzoate (1), is the result of the disappearance of the motive force 045). It has long been knownthat whenan action potential is evoked in characcan cells the streaming halts transiently. The inhibiting effect of the action potential upon streaming has been demonstrated to be due to a momentaryinterrup- tion of the motive force and not to a increase in the viscosity of the cytoplasm 047). As has been stated before, this change is triggered .by transient increase in Ca2+ at the site of motive force generation (49).

METHODOF LATERALCOMPRESSION (87) There is one further approach to measuring the motive force. Whena part of the large cylindrical cell of Nitella showing vigorous cytoplasmic streaming is compressed and flattened betweena pair of parallel flat walls, the two opposedstreamings on the opposite sides of the cell comein contact and fuse. The velocity and the velocity gradient of the fused region of the endoplasmarc changed as the width of the fused region is modified (87). From these measurements it is possible to calculate not only the motiveforce responsible for streaming, but also the force resisting the sliding at the cndoplasm-cctoplasmbound- ary, by the simultaneous flow equation: -F=Rv + */a ~, where F represents

by STEWARD OBSERVATORY on 04/23/06. For personal use only. the motive force, R the sliding resistance per unit velocity, v the marginal velocity, dv/dy the shear rate of the endoplasm, and */a the apparent viscosity of the cndoplasm.For an arbitrary dv/dy, ~a can bc obtained experi- mentally by the data of the isolated endoplasm (85, 87). Wecan measure Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org directly v and dv/dy for each width of the stream. Since both the motive force F and the resisting force R are unknownquantities, we calculate them using two simultaneous flow equations under two different widths of the stream. Again the motive force is found to be 1.7 dyn cm-2, in good agreement with the data previously reported. Further, it is knownthat the major part of the motive force is used in overcomingthe resistance to sliding at the endoplasm-ectoplasmboundary. Since the viscosity of the cell sap is low (1.4-2.0 cp) (unpublished data of N.Kamiya& K. Kuroda), less than of the motive force is used for bringing about its sheafing in the normalcell, Annual Reviews www.annualreviews.org/aronline

228 KAMIYA

Theresistance per unit sliding velocity is knownto be of the order of 230 dyn s cm-3 (87). Recently, Hayashi(51) investigated a theoretical modelof cytoplasmic streamingin Nitella and Charc~On the basis of experimentalresults obtained by Kamiya& Kuroda (81, 82, 85, 87), he derived general theological equations for the non-Newtonianfluid of cytoplasm, constructed a fluid- dynamicmodel of the streaming, derived a general expression for the velocity distribution in the cell, and showedhow the rheological properties of the cytoplasmare estimatedin vivo. He(52) also constructeda theoretical modelof cytoplasmicstreaming in a boundarylayer where the motive force is supposedto exert directly to cytoplasm, and estimated several importantparameters such as the depth of the boundaryregion, rheologieal constants as well as the maximumvelocity in the boundarylayer, and the magnitudeof motiveforce generatedby a unit length of a single subeortieal fibril. Thedepth of the layer was1.3-2.0/zm, while the viscosity wasa small fraction of that estimatedin the bulk layer, and the maximumvelocity was six or seventimes that of the bulk layer. Themagnitude of the motiveforce was2, ca 0.8 dyn, which can be converted to a sliding force 1.2 dyn/em showinga good coincidence with those obtained by Kamiya& Kuroda(82) and Tazawa(145). Molecular Mechanism of Rotational Streaming It is nowestablished that the subeortieal filaments are composedof bundles of aetin filaments all with the samepolarity over their entire length (97). The streamingoccurs steadily in the formof an endless belt in only one direction alongside these filaments. There is no chancefor the endoplasm to movebackward; the track is strictly "one-way."The sliding force (motive force) responsible for the streamingwas measured to be 1-2 dyn cm-z (82, 87, 145) or 3.6 dyn cm-2 (27).

by STEWARD OBSERVATORY on 04/23/06. For personal use only. The ATPcontent in Nitella cytoplasm, wlaich is around 0.5 mM(38), is already saturated for streaming. Nitella myosinhas been isolated and characterized(95), and it has beensuggested that it resides in the endoplasm

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org (22). It wouldbe of great interest to knowwhat kind of aggregationpattern the Nitella myosinhas in vivo, and what kind of interaction occurs with the subeorticalfibrils to producethe continuouslyactive shear force responsible for this endless rotation. Withthe electron microscope,N. F. Allen (7) has shownputative myosinmolecules with bifurcating ends in the endoplasmof Nitella~ Bradley(19) and Williamson(164) suggestedthat putative Nitella myosinmay link with endoplasmicorganelles. William- son’s observationof the subcortieal fibrils in the perfusedcell (165) shows cogently that the streamingis brought about throughinteraction between cortex-attached F-actin bundles and myosin. Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 229

Nagai & Hayama(119, 120) confirmed the existence of manyballoon- shaped, membrane-boundendoplasmic organelles linked to the bundles of microfilaments under ATP-deficient conditions. These organelles, whose shape and size are diverse, have one or more protuberances in the form of rods or horns (119, 120). Electron-dense bodies 20-30 nmin diameter are located on the surface of these protuberances in an ordered array with a spacing of 100-110 rim. It is these electron-dense bodies which link the endoplasmic organelles to F-actin bundles. The dense bodies on the organelles detach from the F-actin bundles on application of ATP. Filaments 4 mnor less in diameter, and different from F-actin in their ability to react with muscle HMM,are attached to the surface of these protuberances. They are probably the same as the 4 nm filaments observed and presumed to be myosin filaments by Williamson (166) and N. S. Allen (7). Nagai & Hayama suggested that the 20 to 30 nm globular bodies attached to the protuberances of the organelles nmyact as functional units whenthe organelles move along the microfilaments, and that they are composed,at least in part, of the functional head of myosin or myosin aggregates, possibly with some other unknownprotein(s). There are several contemporary views regarding the mechanismof rotational streaming other than the above, views which are instructive and suggestivein some respects. But taking into consideration thefacts so far known,the most plausible and rcsonablc picture of thestreaming in NiteIIa is as follows.The rotational streaming is caused by theunidirectional sliding forceof thecndoplasmic organclles loaded with myosin along thc stationary subcorticalfibrils composed of F-actinhaving the same polarity. The motive force,which is theactive shearing force, is produced through the interaction of themyosin with the F-actin on thecortex. Endoplasmic organcllcs, with whichNiteIla myosin is associated,presumably cffcctivcly help drag the rest of theendoplasm so thatthe whole cndoplasmic layer slides alongside the

by STEWARD OBSERVATORY on 04/23/06. For personal use only. cortexof thecell as a mass.Important problems left to bc solvedarc the molecularmechanism of theactive shearing, and its control.

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org CONCLUDING REMARKS

In the foregoing pages, we reviewed molecular and dynamic aspects of cytoplasmic streaming and related phenomenain the two representative materials, myxomyceteplasmodium and characean cells. These two types of streaming both utilize an actomyosin-ATPsystem for their force output, but their modesof movementare quite different. The streaming in Physarum plasmodium is a pressure flow, caused by contractions of actomyosinfilament bundles, while streaming in Nitella is broughtabout by theshearing forceproduced at the interfacebetwcen Annual Reviews www.annualreviews.org/aronline

230 KAMIYA

subcortical actin bundles and putative myosin attached to the endoplasmic organelles. In spite of extensive biochemical studies on contractile and regulatory proteins, and of morphological studies on contractile structures, there has been so far no direct evidence to show that the contraction- relaxation cycle of the slime mold operates on the basis of a sliding filament mechanism. Nor is there any convincing evidence that the changes in aggregation patterns on the part of the microfilaments produce the tension force. There is a possibility that entanglement and network formation are the visual manifestations of the events accompanying the sliding of antiparallel microfilaments against each other, the sliding being effected by myosin dimers or oligomers in the presence of Mg2+-ATPand regulatory proteins. Another important feature of the streaming in the acellular slime mold is its rhythmicity. Wohlfarth-Bottermann discussed four possibilities as a source of oscillation (172). As described before, various physical and chemical parameters of the streaming change with the same period in association with the periodic force production. The origin of oscillation is, however, still unsown. Inrotafiona! streamins in ~Vitdla, the site of themotive force and its mode of action appear to be quite well known now. Probably the sliding mechanism of the cytoplasmic streaming in Nitella is similar in principle to the mechanism of less organized types of streaming in plant cells and saltatory particle movement. But it is not known yet how far the active shear mechanism is applicable to other systems like the myxomycctc plasmodium or amoeba. The basic mechanism of actomyosin-based movement may be common in many different kinds of calls, from muscle to amoeba to plant cells. Our knowledge is, however, still too meager to obtain a unified concept of a variety of cytoplasmic streaming patterns in terms of their molecular organization. by STEWARD OBSERVATORY on 04/23/06. For personal use only. Literature Cited 1. Abe,S. 1964.The effect ofp-chloromer- tion of slime mold myosinand slime curibenzoate on rotational protoplas- moldaetin. Biochemistry8:4976-88 Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org mic streaming in. plant cells. Proto- 5. Allen, N. S. 1974. Endoplasmicfila- plasma 58:483-92 mentsgenerate the motiveforce for ro- 2. Achenbach, U., Wohlfarth-Botter- tational streamingin Nitella. J. Cell mann, K. E. 1980. Oscillating con- Biol. 63:270-87 tractions in protoplasmic strands of 6. Allen, N. S. 1976.Undulating filaments Physarutn-J. Exp. Biol. 85:21-31 in Nitella endoplasmand motiveforce 3. Adelman,M. R., Taylor, E. W. 1969. Isolation of an actomyosin-likeprotein generation. See Ref. 31, pp. 613-21 complex from slime mold plasmodium 7. Allen, N. S. 1980. Cytoplasmicstream- and the separation of the complexiato ing and transport in the characeanalga actin- and myosindikefractions. Bio- Nitellc~ Can.J. Bot. 58:786-96 chemistry 8:4964-75 8. Allen, N. S., Allen, R. D. 1978. Cyto- 4. Adelman,M. R., Taylor, E. W. 1969. plasmicstreaming in green plants, dnn. Further purification and characteriza- Rev. Biophyg Bioeng. 7:497-526 Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 231

9. Allen, R. D. 1977. Concluding remaxks. protoplasmic fibrils in cytoehalasin B Syrup. Mol. Basis Motility. In Interna- treated Nitella rhizoid, Protoplasma tional Cell Biology 1976--1977,ed. B. R. 77:427-35 Bdnkley, K. R. Porter, pp. 403-6. New 22. Chen, J. C. W., Kamiya, N. 1975. Lo- York: Rockefeller Univ. Press. 694 pp. calization of myosin in the internodal 10. Allen, R. D., Allen, N. S. 1978. Cyto- cell of Nitella as suggested by differen- plasmic streaming in amoeboid move- tial treatment with N-ethylmaleimide. ment. Ann. Rev. Biophyx Bioeng. Cell Struet. Funct. 1:1-9 7:469-95 23. Clarke, M., Spudich, J’. A. 1977. Non- 11. Allen, R. D., Kamiya, N., eds. 1964. muscle contractile proteins: The role of Primitive Motile Systern~ in Cell Biology. actin and myosin in cell motility and NewYork: Academic. 642 pp. shape determination. Ann. Rev. Bio- 12. Allen, R. D., Pitts, W. R., Speir, D., cher~ 46:797-822 Brault, J. 1963. Shuttle streaming: Syn- 24. Crooks, R., Cooke, R. 1977. Tension chronization with heat production in generation by threads of contractile slime mold. Science 142:1485-87 proteins. £ Gen. Physiol. 69:37-55 13. All6ra, A., Beck, R., Wohlfarth-Botter- 25. D’Haese, J., Hinssen, H. 1975. Kon- mann,K. E. 1971. Weitreichende, fibril- traktionseigensehaften yon isolierten l~re Protoplasmadifferenzier~ngen und Sehleimpilzactomyosin. 1. Verglei- ihre Bedeutung flit die Protoplasma- ehende Untersuchungen an Fadenmo- str6mung.VIII. Identilizierung der Plas- dellen aus nafiirliehen, rekombinierten mafilamente yon Physarum polyce- und hybridisiert~Actomyosinen yon p/alum als F-Actin dutch An/agerung Schlcimpilzund Mnskcl,Protoplasma von heavy meromyosinin situ. Cytobi- ologie 4:437-49 95:273-95 14. Alldra, A., Wohlfarth-Bottermann, K. 26. D’Haese, J., Hinssen, H. 1979. Aggre- E. 1972. Weitreiehendeflbrillfire Proto- gation properties of nonmuselemyosin. plasmadifferenzierungen und ihr¢ See Ref. 36, pp. 105-18 Bedeutung flit Protoplasmastr~mung, 27. Donaldson, I. G. 1972. Cyclic 10n- IX. Aggregationszusfiinde des Myosins gitudinal fibrillar motion as a basis for mad Bedingungen zur Entstehung yon steady rotational protoplasmic stream- Myosirflilamenten in den Plasmodien ing. J. Theor. Biol. 37:75-91 von Physarumpolflcephalum. Cytobiolo- 28. Dryl, S., Zurzycki, J., eds. 1972. Motile gie 6:261-86 Systems of Ceils (Acta Protozool. 11). 15. Beck, R., Hinssen, H., Komniek, H. Warszawa: Neneki Inst. 418 pp. 1970. Weitreiehende, tlbrillbxe Proto- 29. Ettienne, E. 1972. Subcellular localiza- plasmadifferenzierungen und ihr¢ tion of calcium repositories in plas, Bedeutung fiir die Protoplasma- modia of the aeellular slime mold str~imung. V. Kontraktion, ATPase- Physammpolycephalur~ J. Cell Biol. Aktivifiit und Feinstructur isolieter 54:179-84 tomyosin-Ffiden yon Physarum poly- 30. Fleiseher, M., Wohlfarth-Bottermarm, cephalura. Cytobiologie 2:259-74 K. E. 1975. Correlation between tension 16. Berridge, M. J., Rapp, P. E. 1979. A force generation, fibrillogenesis and ul- comparative survey of the function, trastrueture of cytoplasmic aetomyosin

by STEWARD OBSERVATORY on 04/23/06. For personal use only. mechanismand control of cellular oscil- during isometric and isotonic con- lators. J. Exp. Biol. 81:217-79 tractions of protoplasmic strands. 17. Braatz, R. 1975. Differential histo- Cytobiologie 10:339-65 chemical localization of calcium and its 31. Goldman, R., Pollard, T., Rosenbaum, relation to shuttle streaming in Physa- J. 1976. Cell Motility. NewYork: Cold Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org rum. Cytobiologie 12:74-78 Spring Harbor Lab. 1373 pp. 18. Braatz, R., Komnick, H. 1970. Histo- 32. G~Stz yon Olenhusen, K., Wohlfarth- chcmischer lqachweis ¢ines Bottermarm, K, E. 1979, Evidence of pumpenden Systems in Plasmodien yon aetin transformation during the con- Schleimpilzen. Cytobiologie 2:457-63 traction-relaxation cycle of cytoplasmic 19, Bradley, M. O. 1973, Microfilaments actomyosin: Cycle blockade by phalloi- and cytoplasmic streaming: Inhibition din injection. Cell Tissue of streaming with cytoehaiasin, Z Cell 196:455-70 Sc~ 12:327-43 33. Hasegawa, T., Takahashi, S., Hayashi, 20. Britz, S. J. 1979. Cytoplasmicstreaming H., Hatano, S. 1980. Fragmin: A cal- in Physarurr~ See Ref. 48, pp. 127-49 cium ion sensitive regulatory factor on 21. Chert, J. C. W. 1973. Observations of the formation of actin filaments. protoplasmic behavior and motile chemistry 19:2677-83 Annual Reviews www.annualreviews.org/aronline

232 KAMIYA

34. Hatano,z+ $. 1970.Specific effect of Ca Plant Physiology, NewSer. 7. Berfin/ on movementof plasmodial fragment Heidelberg/NewYork: Springer. 731pp. obtained by caffeine treatment. ~xp. 49. Hayama,t., Shimmen, T., Tazawa, Cell Rex 61:199-203 M.1979. Participation of CAz+ in cessa- 35. Hatano,S. 1973. Contractile proteins tion of cytoplasmicstreaming induced from the myxomycete plasmoOium. by membraneexcitation in Characeae Adv. Biophy~5:143-76 internodal cells. Protoplasma 99: 36. Hatano,S., Ishikawa,H., Sato, H., eds. 305-21 1979. Cell Motility: Moleculesand Or- 50.- Hayama,T., Tazawa,M. 1980. Ca2÷re ganization. Tokyo:Univ. TokyoPress. versiblyinhibits active rotation of ehlo- 696 pp. roplasts in isolated cytoplasmicdroplets 37. Hatuno, S., Matsumura,F., Hascgawa, of Chara~Peotoplasma 102:1-9 T., Takahashi,$., Sato, H., Ishikawa, 51. Hayashi, Y. 1980. Fluid-dynamical H. 1979. Assemblyand disassemblyof study of protoplasmic streaming in a F-actin filaments in Physarumplas- plant cell. Z Theor. Biol. 85:451-67 modiumand Physarum actinin. See 52. Hayashi, Y. 1980. Theoretical study Ref. 36, pp. 87-104 of motiveforce of protoplasmicstream- 38. Hatuno,-S~.., Nakajima,H. 1963. ATP ing in a plant cell. J. Theor. Bio£85: content and ATP-dephosphorylating 469-80 activity of Nitella~ dug Rep. Sc~Works 53. Hepler,P. K., Palevitz, B. A. 1974.Mi- Fae. Sc£ Osaka Univ. 11:71-76 crotubules and microfilaments. Anr~ 39. Hatano, S., Oosawa,F. 1966. Extrac- Rev. Plant Physiol. 25:309-62 tion of an actin-like protein fromthe 54. Higashi-Fujime,S. 1980. Active move- plasmodiumof a myxomyceteand mentin vitro of bundles of mierofila- mteraction with myosinA from rabbit merits isolated fromNitella cell. Z Cell striated muscle.J. CellPhysiol.68:197- Biol. 87:569-78 202 55. Hinssen, 14. 1970. Synthetisehe Myo- 40. Hatano,S., Oosawa,F. 1966.Isolation sinfflamente yon Sehleimpilz-Plas- and characterization of plasmodiumac- modien.Cytoblologie 2:326-31 tin. Biochim.Biophyx dcta 127:489-98 56. Hinssen, H. 1979. Studies on the 41. Hatano, S., Owaribe, K. 1976. Actin polymerstate ofactin in Physarumpoly- and actinin from myxomyceteplas- cephalur~See Ref. 36, pp. 59-85 modia. See Ref. 31, pp. 499-511 57. Hinsen, H., D’Haese, J. 1974. Fila- 42. Hatano, S., Owaribe, K., Matsumura, ment formation by slime mouldmyosin F., Hasegawa,T., Takahashi,S. 1980. isolated at lowionic strength. J. Cell Characterizationof actin, actinin, and Sc£ 15:113-29 myosinisolated from Physaru~Cag Z 58. Hinsen, H., D’I-Iaese, J. 1976. Syn- BoL 58:750-59 thetic tibrils from Physarum ac- 43. Hatano,S., Takahashi,K. 1971.Struc- tomyosin--Selfassembly, organization ture of myosinA from the myxomycete and contraction. Cytobiologie Psallasmodiumt concentrations.and its aggr¢gatiunJ. Mechanochem.at low 13:132-57 Cell Motil. 1:7-14 59. Hitchcock, S. E. 1977. Regulation of motility in nonmusclecells. Z Cell BioZ

by STEWARD OBSERVATORY on 04/23/06. For personal use only. 44. Hatano, S., Takeuchi, I. 1960. ATP content in myxomyceteplasmodium 74:1-15 andits levels in relation to someexter- 60. Inofie, S., Stephens,R. E. 1975.Mole- nal conditions. Protoplasma52:169-83 cules and Cell Movemen~New York: 45. Hatano,S., Tazawa,M. 1968. Isolation, Raven. 450 pp. Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org purification and characterization of 61. Isenberg, G., Wohlfarth-Bottermarm, myosin B from myxomycete plas- K. E. 1976.Transformation of cytoplas- modium. Bioch#n. l~iophys. Acta mic actin. Importancefor the organiza- 154:507-19 tion of the contractile gel retieulumand 46. Hatano, S., Totsuka, T., Oosawa,F. the contraction-relaxationcycle of cyto- 1967. Polymerization of plasmodium plasmic actomyosin. Cell Tissue Re~ aetin. 2~iochirr~ Biophy~ ,~cta 173:495-528 140:109-22 62. Jahn, T. L., Bovee,E. C. 1969.Proto- 47, Hato, B., Ueda, T., Kurihara, K,, plasmic movements within cells. Kobatake,Y. 1976. Phototaxis in true PhysioL Rev. 49:793-862 slime mold Physarumpolycephalurr~ 63. Jarosch, R. 1956. PlasmastrSmungund Cell Struet. Func~1:269-78 Chloroplastenrotation bei Charaeeen. 48. Hanpt,W., Feinleib, M.E. 1979.Physi- Phyton 6:87-107 ology of MoveraentzEncyclo2~edia of 64. Jarosch, R. 1958. Die Protoplasmati- Annual Reviews www.annualreviews.org/aronline

CYTOPLASMIC STREAMING 233

brillen dcr Characecn. Protoplasraa Contractile properties of the slime mold 50:93-108 strand. Acta l~otozool. I hi 13-24 65. Jarosch, R. 1976. DynaraischesVer- 81. Kamiya,N., Kuroda,K. 1956. Velocity halten der Actinfibrillenyon Nitella auf distribution of the protoplasmicstream- Grund schneller Filament-Rotation. ing in Nitella cells. Bot. Mag. Biocherr~Physiol. Pflan~170:111-31 69:544-54 66. Kamitsubo, E. 1966. Motile proto- 82. Kamiya, N., Kuroda, K. 1958. Mea- plasmicfibrils in cells of eharaceae.II surement of the motive force of the Linearfibrillar structureand its bearing protoplasmicrotation in Nitella. Prow- on protoplasmic streaming. Proc. Jp~ plasma 50:144-48 Acad. 42:640-43 83. Kamiya,N., Kuroda, K. 1958. Studies 67. Kamitsubo,E. 1972. A ’windowtech- on the velocitydistribution of the proto- nique’for detailedobservation of chara- plasmic streaming in the myxomycete cean cytoplasmicstreaming. Exp. Cell plasmodium.Protoplasma 49:1-4 Res. 74:613-16 84. Kamiya, N., Kuroda, K. 1964. Me- 68. Kamitsubo,E. 1972. Motitc protoplas- chanical impactas a meansof attacking mic fibrils in cells of the Characeae. structural organizationin living cells. Protoplasma74:53-70 Ann. Rep. Sc£ Works Fac. Sc£ Osaka 69. Kamitsubo, E. 1980. Cytoplasmic Univ. 12:83-97 streaming in eharaceancells: role of 85. Kamiya, N., Kuroda, K. 1965. Rota- subcortical fibrils. Can. J. Bot. tional protoplasmicstreaming in Aritella 58:760-65 and somephysical properties of the en- 70. Kamiya,N. 1953. The motiveforce re- doplasm.Proc. 4th Int. Congr.Rheology sponsiblefor protoplasmicstreaming in Part 4, Syrup. Biorheo£, pp. 157-71. the myxomycete plasmodinm. Ann. New York: Wiley Rep. Sc£ WorksFac. Sc£ OsakaUniv. 86. Kamiya, N., Kuroda, K. 1965. Move- 1:53-83 ment of the myxomyeeteplasmodium. 71. Kamiya,N. 1959. Protoplasmicstream- I. A studyof glycerinatedmodels. Proc. ing. Protoplasmatologia8, 3 a. Vienna: Jpn. Acad. 41:837-41 Springer. 199pp. 87. Kamiya,N., Kuroda, K. 1973. Dynam. 72. Kamiya,N. 1960. Physics and chemis- ics of cytoplasmicstreaming ~ a plant try of protoplasmic streaming. Ann. cell. Biorheology10:179-87 Rev. Plant Physiol. 11:323-40 88. Kamiya,N., Yoshimoto,Y. 1972. Dy- 73. Kamiya,N. 1962. Protoplasmicstream- namiccharacteristics of the cytoplasm ing. Handb.Pflanzenphysiol. 17(2): --A study on the plasmodialstrand of 979-1035 a myxomyeete.In Aspects of Cellular 74. Kamiya, N. 1968. The mechanismof and Molecular Physiology, ed. K. cytoplasmic movement in a myx- Hamaguchi,pp. 167-89. Tokyo:Univ. omyceteplasmodium. In Aspects of Cell TokyoPress, 257 pp. Motility. Syrup.Soc. Exp.Biol. 22, pp. 89. Kamiya, N., Yoshimoto, Y., Mat- 199-214. Cambridge:Cambridge Univ. sumura,F. 1981. Physiolo$icalaspects Press of aetomyosin-basedcell motility. In In- 75. Kamiya,N. 1970. Contractile proper- ternational Cell Biology1980-198L ed. by STEWARD OBSERVATORY on 04/23/06. For personal use only. Acad.ties of 46:1026-31 the plasmodialstrand. Proc. Jpn. H. G. Sehweiger,pp. 346-58. Heidel- berg/NewYork: Springer 76. Kamiya,N. 1973. Contractile charac- 90. Kamiya, R., Maruyama, K., teristics of the myxomycetepins- Kawamura,M., Kikuchi, M. 1972.Mg-

Annu. Rev. Plant. Physiol. 1981.32:205-236. Downloaded from arjournals.annualreviews.org modium.Proc. 4th lng Biophyz Congr. polymerofactin formed under.the influ- enceof fl-aetinin. Biochim.Biophy~ MoMOScOW)tility, pp.Syrup. 447-65 III Biophysics of Acta 256:120-31 77. Kamiya, N. 1977. Introductory re- 91. Kato, T. 1979. Ca2+ uptake of Physa- marks.Syrup. Mol. Basis Motility. See rummierosomal vesicles. See Ref. 36, gel. 9, pp. 361-66 pp. 211-23 78. Kamiya,N. 1979. Dynamicaspects of 92. Kato,2+- T., Tonomura,Y. 1975. Ca movementin the myxomyceteplas- sensitivity of actomyosinATPase puri- modium.See Ref. 36, pp. 399-414 fied from Physarumpolycephalum. J. 79. Kamiya,N., Abe, S. 1950. Bioclectde Biochem. 77:1127-34 phenomenain the myxomyceteplas- 93. Kato, T., Tonomura,Y. 1975. Physa- modiumand their relation to protoplas- rum tropomyosin-troponin complex. mic flow. Colloid Sc~ 5:149-63 Isolation and properties. J.. Biochera. 80. Kamiya,N., Alien, R. D., Zeh, R. 1972. 78:583-88 Annual Reviews www.annualreviews.org/aronline

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94. Kato, T., Tonomura, Y. 1977. Uptake surface of Physarura poly~ephalum. of calcium ion into microsomesisolated Protoplasma 91:107-13 from Physarum polycephalum. J. Bio- 110. Maruyama, K., Kamiya, R., Kimura, chen~ 81:207-13 S., Hatano, S. 1975. fl-Actinin-like pro- 95. Kato, T., Tonomura,Y. 1977. Identifi- tein from plasmodium, d. Biochem. cation of myosin in Nitella flexilig Z 79:709-15 Biocher~ 82:777-82 111. Matsumura, F., Hatano, $. 1978. Re- 96. Kawamura, M., Nagano, K. 1975. A versible superprecipitatinn and bundle calcium ion-dependent ATPpyrophos- formation of plasmodinm actomyosin. phohydrolase in Physarura polyceph- Biochim. Biophys. Acta 533:511-23 alum. Biochim. Biophy~ dcta 112. Matsumura, F., Yoshimoto, Y., Ka- 397:207-19 miya, N. 1980. Tension generation by 97. Kersey, Y. M., Hepler, P. K., Palevitz, actomyosin thread from a non-muscle B. A., Wessdls, N. K. 1976. Polarity of system. Nature 285:169-71 actin filaments in Chaxacean algae. 113. Matthews, L. M. 1977. Ca*+ regulation Proc. Natl./lcad. Sc£ US/I 73:165-67 in caffeine-derived microplasmodia of 98. Kersey, Y. M., Wessells, N. K. 1976. Physarum polycephalum. J. Cell Biol. Localization of actin filaments in inter- 72:502-5 nodal cells of characean algae. J. Cell 114. Nachmias, V. T. 1972. Filament forma- Biol. 68:264-75 Proc.tion byNatl. purified dcad. ScLPhysarum USA 69:2011-14 myosin. 99. Komnick, H., Stockem, W., Wohlfarth- Bottermann, K. E. 1973. Cell Motility: 115. Naehmias, V. T. 1974. Properties of Mechanism in protoplasmic streaming Physarura myosin purified by a potas- and ameboid movement. Int. Rev. sium iodide procedure. Z Cell BioL Cytol. 34:169-249 62:54-65 lOO. Korn, E. D. 1978. Biochemistry of ac- 116. Nachmias, V. T. 1979. The contractile tomyosin-dependenteel1 motility (a re- proteins of Physarum polyeephalum view). Proc. NatL Acad. Sc£ USM and actinpolymerization in plasmodlal 75:588-99 extracts. See. Ref. 36, pp. 33-57 101. Kriiger, J., Wohlfarth-Bottermann, K. 117. Nachmias, V. T., Ash, A. 1976. A calci- ~ 1978. Oscillating contractions in um-sensitive preparation from Physa- protoplasmic strands of Physarum: rum polycephalutr~ Biochemistry 15: 4273-78 stretch-induced phase shifts and their 118. Nachmias, V. T., Huxley, H. E., synchronization. J. Interdiscip. Cycle Kessler, D. 1970. Electron microscope Rex 9:61-71 observations on actomyosin and actin 102. Kuroda, K. 1964. Behavior of naked preparations from Physarum ~olyceph- cytoplasmic drops isolated from plant alum, and on their interaction with cells. See Ref. ll, pp. 31-41 heavy meromyosin subfragment from 103. Kuroda, K. 1979. Movement of cyto- muscle myosin, d.. MoLBiol. 50:83-90 plasm in a membrane-free system. See 119. Nagai, R., Hayama, T. 1979. Ultra- Ref. 36, pp. 347-61 structure of the endoplasmicfactor re- 104. Kuroda, K. 1979. Movement of sponsible for cytoplasmic streaming in demembranated slime mould cyto-

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