J. Cell Sci. i 7 ) 655-668 (1975) 655Printed in Great Britain

CYTOPLASMIC STREAMING IN CHARA:

A CELL MODEL ACTIVATED BY ATP

AND INHIBITED BY CYTOCHALASIN B

R. E. WILLIAMSON*Botany School, Downing St, Cambridge, CBi 3EA, England

SUMMARY

After vacuolar perfusion of Cliara internode cells, the cytoplasm remaining in situ can bereactivated by ATP to give full rates of streaming. Observations during both perfusion andreactivation indicated that the generation of the motive force was associated with fibres con-sisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmicorganelles were immobilized along such fibres. When ATP was introduced, organelles movedalong the fibres at speeds up to 50 /tm s~', but were progressively released from contact to leavethe fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from thefibres without supporting movements. Motility required millimolar Mg2+ levels, free Ca*+ atio~7 M or less and was inhibited by high levels of Cl~ and by pH's on either side of 7-0. Thereactivated movements were rapidly and completely inhibited by 25 fig ml"1 cytochalasin B.The results are interpreted in terms of actin filaments in the stationary cortex interacting witha myosin-like protein which is able to link to endoplasmic organelles. Movement results froman active shear type of mechanism.

INTRODUCTION

The endoplasm of characean algae streams at 40 /tm s- 1 or more. Bundles of micro-filaments situated at the boundary between the stationary cortical cytoplasm and theflowing endoplasm are believed to have a role in the production of the motive forcefor streaming (Nagai & Rebhun, 1966; Pickett-Heaps, 1967; Bradley, 1973). Micro-tubules are not found in positions from which they could contribute to the motiveforce for streaming (Nagai & Rebhun, 1966) and depolymerizing them with colchicinedoes not inhibit the streaming (Pickett-Heaps, 1967).

Three types of study have contributed to our knowledge of the microfilaments andof the way in which streaming is driven. In light-microscope studies (Kamitsubo,1966,1972), fibres have been observed whose position and orientation strongly suggeststhat they are the microfilament bundles. In areas where streaming was recovering fromdamage by centrifugation, a relationship was evident between the reformation of fibresand the resumption of streaming. Organelle movements closely followed theirregular path of the newly formed fibres and occurred only in close proximity tothem. More recently it has been suggested (Allen & Allen, 1972; Allen, 1974) thatthe fibres seen by Kamitsubo - which are fixed, rigid structures - serve only a skeletal

• Present address: Department of Botany, La Trobe University, Bundoora, Victoria 3083,Australia.

42-2

656 R. E. Williamson

function as an anchor for other fibres which project into the endoplasm. Allen believesthat streaming is the result of the propagation of waves of bending along these endo-plasmic fibres.

A second type of study has used the inhibitor cytochalasin B. Many of the processesinitially shown to be sensitive to the inhibitor were thought to be dependent on thefunctioning of microfilaments, and in many cases the microfilaments were disruptedby cytochalasin (Wessels et al. 1971). The inhibition of streaming in intact characeancells (Wessels et al. 1971; Williamson, 1972; Bradley, 1973) and of motility in isolatedcytoplasmic fragments (Williamson, 1972) was consistent with the view that cyto-chalasin in some way affected microfilament functioning. However, many cases arenow known in which cytochalasin inhibits specific membrane-transport systems(references in Pollard & Weihing, 1974). The desire to explain all effects of cyto-chalasin in terms of a single site of action has led to a tendency to regard the plasmamembrane as the primary site of action for the inhibitor. The effects on microfilamentscould then be due to resultant alterations in the ionic composition of the cytoplasm(Estensen, Rosenberg & Sheridan, 1971) or to the disruption of links between mem-branes and microfilaments (Spooner, 1973; Hepler & Palevitz, 1974).

The third approach has involved the demonstration that filaments from twocharacean species (Nitella flexilis: Palevitz, Ash & Hepler, 1974; Chora corallina:Williamson, 1974) react with subfragments of muscle myosin to produce arrowheadfilaments. This indicates strong similarities between the algal filaments and actin(see references in Williamson, 1974). Both papers furnished circumstantial evidencethat the actin filaments were components of the microfilament bundles and definitiveevidence is provided by the in situ decoration of the microfilaments of a glycerinatedcell (unpublished, but quoted by Palevitz et al. 1974).

A further approach to studying problems of motility involves the use of cell models(see Arronet, 1973, for review). These are systems in which the structures responsiblefor motility are preserved in an organized but inactivated state while the cell is madepermeable by chemical or mechanical disruption of the plasma membrane. Theconditions controlling the operation of the motile cell components (energy source,ionic environment, etc.) can then be defined by experiments to restore motility to themodel. Glycerinated muscle fibres which contract in the presence of ATP and Ca2+

ions are perhaps the most familiar example of a cell model. Such models are also ofvalue in separating inhibitors of the in vivo motile process into those affecting directlythe contractile elements, which also inhibit the model, and those inhibiting motilitysecondarily by, for example, interference with the energy supply. The latter type arenot inhibitory to the operation of the model (see Arronet, 1973, p. 48). The only appli-cation of such methods to green plant cells seems to have been the glycerination ofAcetabularia calyculus (Takata, 1961), where the addition of 1 DIM ATP with 1 mMCa or Mg salts was reported to restore transient streaming.

Several studies of streaming in characean cells have employed the technique ofvacuolar perfusion (Kamiya & Tazawa, 1966; Tazawa & Kishimoto, 1964, 1968;Tazawa, 1968; Donaldson, 1972). In such experiments both ends of the cell are cutoff in conditions which permit streaming to continue unimpaired. Experimental

Cytoplasmic streaming in Chara 657

solutions can then be passed through the large central vacuole under the influence ofa small pressure gradient. The flowing solution carries with it much of the endoplasm.In the present study of Chara, attention has been concentrated on that fraction ofcytoplasm not washed out with the perfusing solution and which has been shown toconstitute a cell model. Observations have been made on the role of the microfilamentbundles in streaming, the nature and control of their interactions with endoplasmicorganelles, and of the effects of cytochalasin B on such interactions. The observationsare interpreted in the light of the presence in these cells of actin filaments (Williamson,]974)-

MATERIALS AND METHODSPerfusion. The apparatus (Fig. 1) was designed to allow the process of perfusion to be

observed with an oil-immersion objective. The principles of the method were exactly similarto those of previous studies (references in the Introduction).

To syringe

Perfusionsolut ion^

Objectivelens —

Openedcell

To syringe

Glass" n n g

Microscopeslide

Fig. 1. The perfusion apparatus.

An internodal cell 40—70 mm in length was blotted and placed on a microscope slide. Itsends were sealed with grease into 2 glass rings (16 mm diameter, 9 mm tall), each with a groovein its base through which the cell could pass without damage. After about 60 s the rings werefilled with perfusion solution and the central part of the cell covered with liquid paraffin. Asfound by Tazawa (1968), the organization of the cell was preserved more successfully withliquid paraffin than with an isotonic aqueous solution. Intact Chara cells continue to streamfor several weeks when immersed in liquid paraffin (unpublished results). A coverslip sup-ported at each corner with a small amount of grease was placed over the central portion of thecell.

After placing the assembled apparatus on the stage of the microscope, the levels of fluid inthe 2 rings were equalized by eye. By inserting scissors into the rings, the 2 ends of the cellwere successively removed. Any slight flows of perfusion fluid could be seen by observing themovements of the vacuolar bodies with a low-power objective. Solution was removed witha syringe from the appropriate ring until the vacuolar bodies were being swept with theendoplasm in the normal manner.

Solutions. Sucrose was found in early experiments to prolong streaming against the flow ofthe perfusion solution and to improve the subsequent preservation of the chloroplasts. It wastherefore used with K+ (added either as KC1 or as K2EGTA-ethyleneglycol bis-tetra-aceticacid) as the main osmotic component of the solutions. These were approximately isotonicwith 350 mM sucrose. The level of free Ca3+ ions was controlled with EGTA, which has amuch higher affinity for Caa+ than for Mg=+ and can therefore be used to give low, bufferedlevels of free Cas+ ions (Portzehl, Caldwell & Ruegg, 1964). Solutions were prepared to giveknown levels of free Caa+ and Mg1+ ions, allowing for the binding of both ions to EGTA and

658 R. E. Williamson

ATP. Solutions containing ATP were prepared to have the same level of free Ca1+ and Mg2+

ions as the ATP-free solution they replaced.Cytochalasin B (Imperial Chemical Industries, Pharmaceutical Division) was dissolved at

5 mg ml"1 in dimethyl sulphoxide.Cells. Chara corallina was grown in the laboratory, rooted either in mud covered with

artificial pond water (1 mM NaCl, o-i mM CaClj, o-i mn KC1) or in agar (Sandan, 1955) witha modified Forsberg medium II (Forsberg, 1965).

Microscopy. A Zeiss Universal Research Microscope with differential interference-contrastoptics was used for all observations.

RESULTS

As found in previous applications of the perfusion technique, streaming continuedwithout significant alteration after the ends of the cell had been removed. Thecytoplasm remained as a thin sleeve around the large, central vacuole.

Perfusion. The applied pressure difference when solution was removed from oneof the rings (about 7 mm of perfusion fluid) caused a rapid flow of perfusion solutionthrough the vacuole which carried with it the bulk of the endoplasm. The directionof this flow was routinely arranged to be opposite to the direction of streaming inthe area being observed. It has been observed previously when the bulk of the endo-plasm is moving passively under the influence of centrifugation (Hayashi, 1957) or ofperfusion (Tazawa, 1968; Donaldson, 1972) that some organelles just beneath thechloroplasts continue to move forwards. These movements were stopped only by theapplications of much larger forces, greater than any applied in the present study.Using the improved optical conditions and the presence of considerable lengths ofclear fibres (Fig. 3), it was possible in the present study to see that these persistentforward movements were closely associated with the surface of the fibres. (It shouldbe noted that these are fibres of the type described by Kamitsubo, that is, situatedjust beneath the chloroplasts and showing no bending movements. No evidence ofendoplasmic fibres of the type described by Allen or of the characteristic file oforganelles indicating their propagation of bending waves has been found in this study.)

The forward-moving organelles seen in cells undergoing perfusion appeared to forma single file along the surface of the fibre. Their forward movements continued evenwhen organelles to the side and beneath were being swept backwards. Members ofthe forward-moving file were from time to time swept away with the backward-flowingendoplasm. The association of the forward movements with the fibres was seenmost clearly when a fibre was oriented obliquely to the main direction of streaming,usually as the result of an abrupt bend (Fig. 2). Organelle movements faithfullyfollowed the deviations of such fibres.

The inactive state. Routinely the cell was first perfused with a solution of salts andsucrose lacking ATP. The exact composition of this solution had, within the limitstested, no major effects on events prior to reactivation (see below). The forward move-ments described above, lasting for periods of up to 60 s, continued until very littleremained of the endoplasm flowing with the perfusion fluid. The forward-movingorganelles then abruptly ceased moving and became anchored to the fibre alongwhich they had been travelling (compare Figs. 3 and 4). In this inactive state (no

Cytoplasmic streaming in Char a 659

added ATP), very few of these organelles could be dislodged from the fibres even byrapid and prolonged perfusion.

Reactivation. The ATP-free solution used for the initial perfusion was removedfrom the 2 glass rings and a solution identical but for the presence of 1 mM Na2ATPwas added to one ring. (The addition was made so that the direction of flow of theATP solution was the same as that of the initial perfusion.) ATP was routinely added60 s after entry into the inactive state. The response of the preparation to ATP had2 components: the resumption of organelle movements and the loosening of thelinkages holding the organelles to the fibres.

The conditions affecting the velocity of the reactivated streaming will be discussedin detail below, but in vivo rates could be obtained immediately after reactivationunder quite a range of the conditions tested. The direction of the reactivated streamingwas always the same as the in vivo direction. The movements were intimately asso-ciated with the fibres; this was seen most clearly where gaps between chloroplastswere spanned by single fibres and where a fibre possessed an abrupt bend (Fig. 2).Organelle movements were confined to the fibre and followed any deviations in itstrack. Movements could involve single organelles or cytoplasmic fragments - thatis, groups of organelles moving as a unit, not necessarily all in contact with the fibre.Under conditions giving high rates of streaming, organelles moved smoothly overdistances greater than 100 /tm. When the movements were slower, the progress of anorganelle was often discontinuous, movements of a few to several tens of micrometresbeing interspersed with periods of Brownian motion near the fibre. The resumptionof active movements was apparently dependent on renewed contact with the fibre.Simultaneous movements of organelles at different rates along the same fibre wereobserved. The maximum duration of motility was about 50 min and the velocity ofthe movements declined during this period. As in the intact cell, no movements ofthe fibres themselves were seen.

The second characteristic response to ATP was the release of organelles from thetight binding to the fibres which characterized the inactive state. Many organellescould be swept away even by very gently flowing solutions containing ATP. For thisreason, ATP was introduced under a very small pressure difference and this wasremoved shortly after reactivation had occurred. The number of organelles under-going movements associated with the fibres declined noticeably with time, more andmore organelles being found free in the central vacuolar space. A consequence of thiswas that the fibres came to have an extremely clean appearance (compare Figs. 5and 6; see also Fig. 2).

If ATP were included in the initial perfusion solution, no condition correspondingto the inactive state was observed. The few organelles remaining near the fibres con-tinued moving and were easily swept away by the perfusion solution to leave the veryclean type of fibre.

Conditions for reactivation. Very low rates of movement were obtained with any ofthe solutions tested at pH 6-o (10 mM morpholinoethane sulphonic acid as buffer).At pH 8-0 (10 mM Tris buffer), no rates higher than 30 /tm s"1 were obtained. Themost thorough study of reactivation was therefore made at pH 7-0 (10 mM piperazine-

660 R. E. Williamson

A^N'-bis^-ethane sulphonic acid). Here the full in vivo rate of streaming(50/tms"1) could be obtained subject to the following conditions: (i) The level offree Ca2+ ions should be io~7 M or less. No evidence of a requirement for Ca2+ ionscould be found, full rates of streaming being obtained even with 50 mM EGTA and noadded calcium, icr6 M Ca2+ produced an inhibition of about 20 %; io~6 M and aboveproduced an inhibition of about 80 %. Qualitatively, the effects of the various Ca2+

levels were similar at pH 8-0; all velocities were, however, lower than underequivalent conditions at pH 7-0. (ii) The level of free Mg2+ ions should be at least1 DIM, which would be in equilibrium with a MgATP level of 0-9 IHM. O-I mM freeMg2+ (o-6 m\i MgATP) was slightly inhibitory, the inhibition being almost totalwith no added Mg. (iii) The level of Cl~ ions should be 80 mM or less, n o mM beingstrongly inhibitory.

Changing the K+ level between 35 and 170 niM (with compensating alterations inthe sucrose concentration) had no significant effect on the velocity of the reactivatedstreaming. More extreme K+ levels were not investigated.

ADP and AMP. With 200 mM sucrose, 50 mM EGTA and 4 mM free Mg2+,streaming was reactivated with a velocity of 50 /*m s"1 by 1 mM ATP. Replacing theATP with 1 mM ADP resulted in velocities not exceeding 11 /urn s"1. AMP (1 and10 mM) produced neither movement nor dissociation of organelles from the fibres.

Pyrophosphate. Ten mM Na4P2O7 was substituted for the adenine nucleotides testedabove. No movements at all resulted, but the progressive release of organelles fromtight binding to the fibres was evident. After some 10-15 min, this left extremely cleanfibres similar to those seen after ATP treatment (see Figs. 2 and 6). A controlexperiment with 20 mM Na2HPO4 produced neither release nor movement of theorganelles. One mM pyrophosphate did not give completely clean fibres.

Cytochalasin B. With the sucrose, EGTA and Mg2+ levels used to test ADP andAMP, cells were perfused with solutions lacking both ATP and cytochalasin. As soonas the inactive state had been reached, perfusion was continued with a solutionidentical but for the presence of cytochalasin B; 60 s after entry to the inactive state,a solution containing both cytochalasin and 1 mM ATP was perfused in the usualmanner for reactivation. With 25 /tg ml"1 cytochalasin, both the release of theorganelles and their movements were almost completely abolished. Movements didnot usually exceed about 20 /tm in total before stopping completely, and the fibresremained heavily coated with organelles; 10/tg ml"1 cytochalasin caused considerablebut incomplete inhibition of both processes. The effects of 25 /tg ml"1 cytochalasinwere partially reversed by subsequent perfusion with a solution containing anidentical level (0-5 %) of dimethyl sulphoxide. As the speeds obtainable on reactiva-tion decline somewhat with time spent in the inactive state, the fact that full rates ofstreaming were not obtainable on washing out the cytochalasin may simply reflectthe extra delay in such experiments.

DISCUSSION

Site of force production. Two observations in this study point to the generation ofthe motive force being intimately related to the microfilament bundles forming the

Cytoplasmic streaming in Char a 661

fibres first described by Kamitsubo. (That the fibres in these particular cells consistof microfilaments has been confirmed by unpublished electron micrographs.) Firstly,in cells undergoing perfusion, organelles close to the fibres continue to move forwardswhile the rest of the endoplasm is moving backwards. In such a situation, forwardmovements must depend on a locally generated force and cannot be explained byforces generated elsewhere and transmitted by the viscosity of the endoplasm.Secondly, in the reactivated cell lacking most of its endoplasm, movements are veryobviously associated with the microfilament bundles. This is most convincing withthe movements of single organelles along the lengths of fibres between well spacedchloroplasts and where such fibres follow an irregular course with bends.

Interaction of organelles and fibres. Two effects of restoring ATP to the Char a modelwere apparent: organelles formerly tightly bound to a fibre moved along it, but showedadditionally an increased tendency to be released from contact with it. A plausibleexplanation of these results can be advanced in terms of actin filaments anchored inthe cortex (Williamson, 1974) interacting with an as yet uncharacterized myosin-likccomponent which can link the actin to endoplasmic organelles.

Thus in the inactive state produced by the absence of ATP, actin and myosin wouldbe in rigor combination, linking the organelles tightly to the fibres. Now ATP hasa dual effect on a muscle in rigor, causing by its binding to myosin the detachmentof the cross-bridges and by its hydrolysis their cyclical interaction with actin to pro-duce movement (see, for example, the paper of Reedy, Holmes & Tregear, 1965). In theChara system the binding of ATP to detach linkages between the fibres and theendoplasmic organelles would tend to release the latter; the hydrolysis of ATP couldpower cyclical movements of the same linkages causing the organelles to move alongthe fibre just as thin and thick filaments move past each other in muscle.

A more critical test of the theory involves separating the effect caused by the bindingof ATP from the effect caused by its hydrolysis. In muscle, this can be done byinhibiting the actomyosin ATPase by removing Ca2+ ions; this results in the releasedbridges remaining detached because the thin filaments are turned off (Reedy et al.1965; Huxley, 1968). With the Chara model, this is ineffective as there is no evidencefor a Ca2+ requirement. A similar effect can also be achieved in muscle by using anon-hydrolysable analogue of ATP; for example, inorganic pyrophosphate causessignificant detachment of the myosin cross-bridges (Lymn & Huxley, 1972). Thisapproach does separate the 2 effects in Chara, allowing detachment to occur in theabsence of any movement. While pyrophosphate is a less-effective dissociating agentthan ATP, inorganic phosphate is completely without effect. This indicates thesignificance of the pyrophosphate linkage irrespective of its capacity to be hydrolysed.

Until Chara myosin is identified and its subcellular location established, the theorycannot be fully tested. We do, however, know that rabbit myosin is dissociated fromChara actin by ATP (Williamson, 1974), so that it would be surprising if Charamyosin were not. The observation that all the arrowheads on a single bundle of actinfilaments point in the same direction (Palevitz et al. 1974) is also consistent with thistheory, as the direction of the arrowheads would specify the direction of myosinmovement and therefore cause unidirectional organelle movements.

662 R. E. Williamson

The theory is a particular application of the 'active shear' mechanisms previouslydiscussed in general terms (Huxley, 1963, 1973; Wolpert, 1965; Jahn & Bovee, 1968).The essential point stressed by these authors was that widely different forms ofmotility could result from the assembly in different ways of proteins essentially similar tothose of muscle. The present study demonstrates for the first time that motile organ-elles can link to the fibres in a way suggesting that a myosin-like protein is involved.

Jons and motility. Organelles released from the fibres by ATP or pyrophosphate candisperse freely in the central vacuolar space of the perfused cell. There can thus beno continuous membrane of the tonoplast type. Indeed the tonoplast must be carriedalong with the perfusing solution in order to transmit the flow of that solution to theendoplasmic organelles. This lack of tonoplast presumably explains the sensitivityto ionic conditions shown by the reactivated streaming when compared to the lackof sensitivity shown by the streaming cytoplasm before full perfusion.

Of the various conditions controlling the reactivation of motility, perhaps the mostinteresting is that the Ca2+ level be kept at or below io~7 M for maximal velocities tobe achieved. This was found to be the case with wide variations in the K+, Mg2+ andsucrose levels, as well as at pH 8-0. This contrasts sharply with the requirements foractin-myosin interaction in muscle (see Weber & Murray, 1973) where interaction isblocked in the resting muscle by the low level of free Ca2+ ions ( < io~7 M). Contrac-tion is then triggered by a rise in the level of free Ca2+ ions. Evidence concerning theregulation of actin-myosin interaction outside muscle is limited, much but not all ofit pointing to regulation by Ca2+ ions (Pollard & Weihing, 1974). It may be notedthat Ca2+ sensitivity is not intrinsic to actin-myosin interaction but is conferred byadditional polypetides associated with either the actin or the myosin component(see Weber & Murray, 1973). Control might therefore be expected to show morevariation than the basic mechanical process, and this might particularly be thecase in comparing a continuously active system such as streaming in Chara, withsystems showing on-off control such as muscle. Data either on other Chara modelpreparations (glycerol or detergent extracted) or on the Chara actomyosin ATPaseitself will help to establish the significance of the present results.

No data are available on the level of free Ca2+ ions in plant cytoplasm. Total Ca2+

levels in the cytoplasm of Nitella translucens have been reported in the millimolarrange (Spanswick & Williams, 1965) but, as in animal cells, much of this could beretained in membrane-bound organelles (endoplasmic reticulum, mitochondria,vacuoles). The need for millimolar levels of Ca2+ in the perfusion solution reportedin earlier studies with N.flexilis (Tazawa & Kishimoto, 1964) reflects the level neededin the vacuole for prolonged, normal operation of the endoplasm. (Tazawa & Kishi-moto used a gentle perfusion technique to leave the endoplasm intact and functional.)The much lower levels found in the present experiments are likely to be more relevantto the in vivo levels of free Ca2+ in the endoplasm itself.

The conditions under which the model was reactivated appear reasonably physio-logical. Thus characean cytoplasm is generally poor in Cl~ ions, which were foundto inhibit motility at high concentrations, and rich in K+ ions, the main cation usedin the reactivation experiments (see table 1 in MacRobbie, 1970, for a summary of

Cytoplasmic streaming in Chora 663

ionic levels in characean cytoplasm). The total Mg2+ level in the flowing cytoplasm ofthese Chara cells was measured as 3-6 raM (unpublished results). The ATP level inNitella was measured as 0-04 mM on a total cell volume basis (Hatano & Nakajima,1963), which would be close to 1 mM if contained exclusively in a cytoplasmic com-partment occupying about 5 % of the total volume.

Energy source. The low rates of movement supported by exogenous ADP may bedue to its conversion to ATP by, for example, an adenylate kinase enzyme in theresidual cytoplasm. The specificity of the presumptive myosin cannot be satisfactorilystudied in the present system.

Cytochalasin B. At 25 /tg ml"1, cytochalasin B causes a rapid and complete inhibi-tion of streaming by ATP. Applied externally to an intact Chara cell, such totalinhibition of movements near the fibres requires periods longer than 60 min, evenwith 50/tg ml^1 (unpublished results). Thus the effectiveness of cytochalasin isreduced if it has to work from outside the cell, suggesting that the plasma membraneis a barrier to inhibition rather than its mediator. (See de Laat, Luchtel & Bluemink,1973, for a similar conclusion in regard to egg cleavage.) Furthermore, inhibition israpid and total even if the energy supply and ionic conditions around the filament-organelle system are regulated as in these reactivation experiments. The observationsstrongly suggest that cytochalasin is exerting a fairly direct effect on the filament-organelle system rather than one mediated by the plasma membrane or by changesin ionic environment.

Following treatment with ATP and cytochalasin, the organelles remain predomi-nantly linked to the fibres, indicating that their ATP-induced dissociation has beenblocked. Careful observation of Chara cells inhibited by externally applied cytochala-sin has revealed that some immobile organelles become attached for long periods tothe fibres. On subsequent perfusion, many of these organelles are not swept awaywith the perfusing solution, but few new ones are added to the fibres in the way thathappens in the uninhibited cell on entry to the inactive state (unpublished results).It may be that cytochalasin both reduces the affinity of the organelles for thefibres and the ability of ATP to dissociate the fibre-organelle linkages. Few linkageswould then form, but those that did would be more stable to dissociation by ATP.A more refined in vitro system will be needed to test these ideas.

Mechanism of streaming. The present results provide experimental evidence for therole of the microfilament bundles in streaming and the first evidence about the natureof their interaction with endoplasmic organelles. The existence of organelle-fibrelinkages which are stable in the absence of ATP is expected only with the activeshear mechanism described here and not with the mechanism dependent on wavepropagation (Allen, 1974). This does not necessarily preclude the operation addi-tionally of a wave propagation system, although theoretical treatments (Donaldson,1972) favour force generation being limited to that narrow zone just beneath thechloroplasts where active shearing would take place. It will obviously be of con-siderable importance to establish whether actin and myosin are confined solely tothis location in the cell, or whether they occur in functional combination throughoutthe endoplasm.

664 R- E. Williamson

I thank Dr E. A. C. MacRobbie and Dr H. E. Huxley for many valuable discussions, andChurchill College for a Junior Research Fellowship, during the tenure of which this work wascarried out.

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Microbiol. 15, 270-293.{Received 25 September 1974)

666 R. E. Williamson

Figs. 2-6. The scale line in each case indicates 5 /tm.Fig. 2. A fibre (arrows) with an abrupt bend. Such bends were of value in con-

firming the role of fibres in motility, organelle movements following all such deviationsof the fibres. Photographed after perfusion with ATP; note the characteristic absenceof associated organelles. Other fibres are just visible beneath the files of chloroplasts.

Cytoplasmic streaming in Chara 667

Fig. 3. Fibre (arrows) in a cell open at both ends but not yet perfused. Endoplasmin situ and still streaming.

Fig. 4. The same area after perfusion with an ATP-free solution. Most of theendoplasm has been swept away, but some organelles and other material can be seenassociated with the fibre (arrows). Comparison of Figs. 3 and 4 indicates that thefibres seen after perfusion correspond to those visible before perfusion.

668 R. E. Williamson

Fig. 5. A fibre (arrows) after perfusion with an ATP-free solution. Note as in Fig. 4the association of organelles with the fibre.

Fig. 6. The same fibre as in Fig. 5, but seen after perfusion with ATP. The fibre,along which organelle movements occurred, became progressively freed of associatedorganelles.