-
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
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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
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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
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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
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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-
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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
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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.
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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.
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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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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.
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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.
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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.