microfilaments and cytoplasmic streaming: inhibition of streaming … · 2005. 9. 6. · 33° m. 0....

J. Cell Sci. 12, 327-343 (i973) 327Printed in Great Britain

MICROFILAMENTS AND CYTOPLASMIC

STREAMING: INHIBITION OF STREAMING

WITH CYTOCHALASIN

M. O. BRADLEYDepartment of Biological Sciences, Stanford University, Stanford, California 94305,U.S.A*

SUMMARY

Cytochalasin B reversibly inhibits cytoplasmic streaming in both Nitella and Avena cells.Colchicine, on the other hand, has no effect on streaming in either plant; nor does colchicineprevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of proteinsynthesis by cycloheximide has no effect on either streaming itself or on the recovery ofstreaming after cytochalasin withdrawal. All this suggests that microfilaments may provide onecomponent of the structure that generates the streaming force and that microtubules playlittle, if any, role in the process.

Ultrastructural studies of Nitella demonstrate that microfilaments are localized at theboundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments areorganized in discrete bundles, with possible cross-bridges between individual filaments in eachbundle. These bundles are closely associated with the extensive endoplasmic reticulum.

Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does insome animal-cell filaments. Since the molecular mechanism of cytochalasin's action is unknown,there may be no necessary correlation between functional inhibition by the drug and alteredmicrofilament morphology.

A model is advanced which proposes that streaming is generated by an interaction betweenmicrofilaments and the endoplasmic reticulum.

INTRODUCTION

Cytoplasmic streaming occurs in a great variety of plant and animal cells (seeKamiya, i960; Allen & Kamiya, 1964, for reviews). The forces that direct suchstreaming are not well understood; however, since cytoplasmic particles do not moveby the action of forces generated by the particles themselves, some external cytoplasmicforces must be moving them (Rebhun, 1967). The fibrillar microtubule and micro-filament systems are possible candidates for this force-generating role and have beenhypothesized as the structural elements driving various types of streaming. Micro-tubules, for instance, have been implicated in 'fast' axoplasmic transport in nervecells (Kreutzberg, 1969), in the streaming and saltatory movements of heliozoanaxopods (Tilney & Porter, 1965; Tilney, 1968), in melanin granule migration in fishmelanophores (Bikle, Tilney & Porter, 1966), in chromosome movement in the mitoticapparatus (Mclntosh, Hepler & Van Wie, 1969), and in higher plant (Ledbetter &Porter, 1963, 1964) and algal (Sabnis & Jacobs, 1967) cytoplasmic streaming. Micro-filament-based motility has been proposed for phenomena such as cytoplasmic

*Address for reprint requests: Department of Medical Microbiology, Stanford UniversitySchool of Medicine, Stanford, California, 94305, U.S.A.

328 M. 0. Bradley

streaming and movement of Amoeba (Pollard & Ito, 1970), Physarum (Wohlfarth-Bottermann, 1964), and Difflugia (Wohlman & Allen, 1968), organelle movement incultured rat embryo cells (Buckley & Porter, 1967), glia, nerve and fibroblast move-ment in cell culture (Wessells et al. 1971), and cytoplasmic streaming in algae (Nagai& Rebhun, 1966), and higher plants (O'Brien & Thimann, 1966; Parthasarathy &Muhlethaler, 1972).

The interpretation linking microfilaments or microtubules to many of thesephenomena is based on a correlation between the observed biological event and thespatial localization of the respective organelle. In some cases both microtubules andmicrofilaments are found in the same locus and so permit alternative explanations fora given phenomenon.

In Nitella, Kamiya & Kuroda (1956, 1963) and Hayashi (1964) have shown byphysical measurements that the motive force for streaming is localized at the interfaceseparating the flowing endoplasm from the stationary cortical gel layer. At this inter-face, Nagai & Rebhun (1966) found large bundles of 5-nm diameter microfilamentsoriented with their long axis parallel to the direction of streaming. They proposed thatthese microfilament bundles generate the motive force for rotational streaming. Micro-tubules, on the other hand, are located just below the plasma membrane in thestationary ectoplasm. Since they are on the opposite side of the stationary chloroplastsfrom the streaming endoplasm, and since they are not necessarily oriented parallel tothe axis of streaming, microtubules are considered less likely to be of importance in thestreaming phenomenon.

The site at which the motive force is generated has not been determined by physicaltechniques in Avena, as it has been for Nitella. Nevertheless, O'Brien & Thimann(1966) found bundles of 5-nm microfilaments in Avena epidermal and parenchymalcells, and hypothesized that Avena streaming also depends upon such filaments. Asin Nitella, the Avena filament bundles parallel the direction of streaming.

Drugs that selectively attack these filamentous organelles can be used to test thevalidity of different hypotheses and to discriminate between microtubule and micro-filament based processes. This paper pursues this approach in an investigation of cyto-plasmic streaming in the alga Nitella and the higher plant Avena by utilizing cytocha-lasin B (Carter, 1967; Wessells et al. 1971) and colchicine (Pickett-Heaps, 1967),drugs that are thought to attack microfilaments and microtubules respectively. Apreliminary report of part of this work has been published before (Wessells et al.1971); this paper extends the previous work and discusses a mechanism for cytoplasmicstreaming.

MATERIALS AND METHODS

PlantsExperiments were performed on Nitella sp. collected from a local pond and on Nitella

axillaris maintained in continuous laboratory cultures (kindly supplied by Dr Paul B. Green).Observations were made on groups of 4 internodes, from 2 to 3 cm long, in sterile filtered pondwater or in glass-distilled water. All experimental dishes were kept on a light tray except duringobservations.

Effect of cytochalasin on streaming 329

Avena sativa seedlings were grown in the dark for 3 days after an initial red light treatment.Outer epidermal sections were cut from the coleoptiles and incubated in previously oxygenated005 M sodium phosphate buffer, pH 7-5, with 1-5% (w/v) sucrose added. Control streamingcontinued for from 10 to 15 h under these conditions.

Streaming measurements

For Nitella, the rates of endoplasmic particle movement were measured with a stopwatchand an ocular micrometer.

For Avena, rates of particle movement were not measured because of great intracellularvariations in rate. Instead, qualitative effects of different drug treatments were assayed by notingwhether streaming in a section was vigorous in all cells, slowing or stopped in some cells, orcompletely stopped in all cells.

Drugs

Cytochalasin B was used at concentrations between 1 /tg/ml (2-1 x 10 6 M) and 30 figjml(63 x io~5 M). The drug is sparingly soluble in water, so stock solutions were prepared indimethylsulphoxide (DMSO) and diluted to the appropriate concentration with medium. Thefinal DMSO concentration in experimental and control cultures was always 1 % (v/v). In rever-sal experiments, cytochalasin was removed by washing the plants with 5 changes of drug-freemedium.

Colchicine (Calbiochem, A grade) was dissolved in medium, at io~2 M final concentration,shortly before use.

Cycloheximide (10 /tg/ml or 3-5 x io~5 M final concentration, Actidione, Upjohn) was addedto the media in order to inhibit protein synthesis before and after recovery from cytochalasintreatment.

Electron microscopy

Nitella internodes, 1 to 2 cm long, were fixed for 15 h at room temperature with a solutionof 3 % glutaraldehyde in 0006 M potassium phosphate buffer pH 7 1 . They were then washed5 times over a period of 30mm with 0025 M potassium phosphate buffer, p H 7 - i . Afterwashing, the internodes were cut into o-5-cm pieces with a razor blade and placed in 2 %osmium tetroxide (buffered to pH 7 1 with 0025 M phosphate buffer) for 1 h at 3 °C. Thesections were washed again with phosphate buffer and then dehydrated through an ethanolseries (15 to 100%) overnight. Following clearing with propylene oxide, embedding was donein Epon. Thin sections were cut on a Sorvall MT-2 ultramicrotome, stained with uranyl acetateand lead citrate (Venable & Coggeshall, 1965) and examined with an Hitachi HU-11E electronmicroscope.

Radioisotope techniques

Uptake. Avena seedlings were grown for 3 days in the dark. Leafless coleoptile sections 13 mmlong were incubated for varying lengths of time in 2 /tCi/ml of L-[4,5-3H]leucine. After incuba-tion the sections were chilled, washed 4 times with unlabelled leucine at io4 times the concen-tration of the labelled leucine (io4 x leucine), chopped into short sections and washed again 4times. The chopped tissue was digested with 3 ml of NCS (Amersham/Searle) for 1 h at 45 °C.Ten millilitres of Bray's (i960) scintillator fluid were added to the digestion mixture and thesamples were counted with a Nuclear Chicago Mark II. Disintegrations per min (dpm) werecalculated by the channels-ratio method.

Incorporation. The Avena sections were treated in the same way as for the uptake studies untilafter the final washing. Then the sections were sonicated for 2 min with a Branson sonicatorin io4 x leucine. The method of Lowry, Rosebrough, Farr & Randall (1951) was used to deter-mine the protein concentration of the sonicate. Cold, 10% trichloroacetic acid (TCA) withio4 x leucine was added to the sonicates for 12 h. This mixture was heated to 90 °C for 30 min,chilled and filtered through Whatman GF/C glass fibre filters with 7 washes of cold 5 % TCAplus io4xcold leucine. Protein was digested from the dried filters with 12 ml of NCS. Thedigests, including the filters, were counted using the procedures outlined above.

33° M. 0. Bradley

Fig. i. The effect of cytochalasin on cytoplasmic streaming in Nitella internode cellsData from 3 typical cells are shown. At the times indicated, 30 /*g/ml of cytochalasin wasadded ( + cb) and later removed ( - cb) by washing the cells with 5 changes of cyto-chalasin-free medium. O—O, control; A—A, 1 % (v/v) dimethylsulphoxide; % %30 /tg/rnl cytochalasin.

RESULTSEffects of cytochalasin on streaming

Cytochalasin B stops cytoplasmic streaming in both Nitella internodal cells (Fig. 1),and in Avena parenchymal cells. Avena sections are slightly more sensitive to the drugthan the Nitella internodes. Thus, Nitella is insensitive at concentrations of 3 /tg/mlor less, while Avena is insensitive below 1 /tg/ml. Avena streaming was stopped within30 min to 1 h by 30 /tg/ml of cytochalasin.

Just before streaming is stopped, cytoplasmic particles in both systems move inparallel short jerks, often at angles oblique to the original streaming axis. The Nitellastream contains a small number of free chloroplasts that spin rapidly around their axis.As the stream is slowed by cytochalasin, spinning of chloroplasts stops, even thoughthey continue to move forward at approximately 5 /tm/s.

If the plants are washed with cytochalasin-free medium, streaming begins againwithin 15-30 min in both plants. In Nitella, the 'recovered' streaming appears normaland the rates attain 95 % or more of the initial values within 5 h; in Avena,' recovered'streaming is vigorous and qualitatively similar to the controls after 3-4 h. Nitellarecovery will occur after as long as 12 h of arrested streaming due to continuous drugtreatment. Although shorter term treatment appears to be harmless, after 24 h in cyto-chalasin Nitella cells begin to degenerate and by 36 h most are dead. This is indicatedby the clumping of the cytoplasm and the randomization of the chloroplast files. Suchlong term cytotoxity could be the result of the drug itself, or it could be a secondaryeffect due to the cessation of streaming.

Effect of cytochalasin on streaming

+ cb

331

Fig. 2. The effect of combinations of cytochalasin, colchicine, and cycloheximide oncytoplasmic streaming in Nitella. Data from 4 typical internodes are shown. At thetimes indicated 30 /tg/ml of cytochalasin was added (+ cb) and later removed (— cb)by washing the cells with 5 changes of their original incubation medium. O—O,io~2 M colchicine; # — 9 , io~2 M colchicine with 30/tg/ml of cytochalasin added andremoved at the indicated times; V —V, 1 o /*g/ml of cycloheximide; V —T, 10 /tg/mlcycloheximide with 30 /tg/ml of cytochalasin added and removed at the indicatedtimes.

Effects of colchicine on streaming

Colchicine was applied to Nitella and Avena cells in order to study the role of micro-tubules in cytoplasmic streaming. Colchicine has no effect upon streaming in Nitellafor at least 24 h (Fig. 2), or in Avena for 10 h.

Possible interactions between microtubules and microfilaments were examined bypermitting cells to 'recover' from cytochalasin treatment in the presence of colchicine.In this experiment Avena sections and Nitella internodes were preincubated for 2 h incolchicine. Then 30/tg/ml of cytochalasin was added until streaming stopped; 1 hlater cytochalasin was washed out and the plants were allowed to 'recover' in thepresence of colchicine alone. Both Nitella (Fig. 2) and Avena streaming 'recover'with the same kinetics as in colchicine-free controls. These findings all suggest thatmicrotubules play an insignificant role in streaming generation.

Effects of cycloheximide on streaming

Cycloheximide, an inhibitor of protein synthesis occurring on polysome-bound80-s ribosomes, was applied to normally streaming plants and to those 'recovering'from cytochalasin. Both Nitella (Fig. 2) and Avena streaming is unaffected by long-term cycloheximide treatment. Furthermore, the drug allows normal 'recovery' fromcytochalasin inhibition of streaming. These results suggest that the proteins of thestreaming apparatus do not turn over rapidly and that the reconstitution of a func-

332 M.O. Bradley

Table i. Effects of cytochalasin and cycloheximide on [3H]leucineincorporation by Avena coleoptiles

Three-day-old, darkgrown, leafless coleoptile sections 13 mm long were incubated for 2 or20 h in 2 fiCi/ml of L-[4,5-3H]leucine. Ten sections were used for each of 3 replicates of oneexperimental treatment. For preparative procedures see Methods.

Labellingtime, h

2

2O

Experiment

ControlCycloheximide,

Dimethylsulphoxide,1 % v/v

Cytochalasin,30 /*g/ml

ControlCycloheximide,

io/^g/mlDimethylsulphoxide,

1 % v/vCytochalasin,

30 //g/ml

* (n) = number

dpm/mgprotein (n)*

665 x io4 (3)9 1 0 X i o 3 (3)

6-31 x io4 (3)

6-50 x io4 (3)

6-68 x io6 (3)1 09 x io5 (3)

5-87 x i o 5 (3)

3-40 x 1 o5 (3)

of experiments performed

0//ochange

- 8 6 3

-5'4°

— 2 6 0

—

-836

— I2-O

-42-2

Probability

< 0001

> 005

> 005

< 0001

> 005

< 0001

tional streaming apparatus after cytochalasin inhibition requires neither immediatelyprior nor concomitant protein synthesis.

The dose of cycloheximide used in these experiments decreased [3H]leucine incor-poration into Avena coleoptiles by 86-3 % in 2 h and by 83-6 % in 20 h as seen in Table1. Because an abundant bacterial microflora adheres to Nitella cell walls, it is impossibleto measure protein synthesis directly in this system. Instead, we determined the effectof cycloheximide on the growth rate of Nitella internodes, making the assumption thatgrowth rate and protein synthesis must be coupled in some way. The results showthat cycloheximide completely stops internode elongation (Fig. 3); this is consistentwith the supposition that the drug also prevents protein synthesis.

Effects of cytochalasin on protein synthesis and growth

Table 1 shows the effect of cytochalasin on short and long term [3H]leucine incor-poration into presumed protein in Avena sections. During a 2-h labelling period, thereis no significant decrease in the number of counts incorporated. However, after 20 hof continuous labelling, total incorporation is decreased by 42 %. Because cytochalasinis applied for only 2 or 3 h in most experiments dealing with effects on streaming, andbecause direct inhibition of protein synthesis with cycloheximide has no effect onstreaming, it is clear that the rapid biological effects of the drug cannot be explainedby decreased rates of protein synthesis.

The appreciable decrease in long term incorporation is not yet understood. Oneexplanation is that the partial cytochalasin inhibition of leucine uptake (if real), seen in


25 -

Fig. 3. The effect of cycloheximide and cytochalasin on elongation of Nitella inter-nodes. The initial length of every internode between 2 and 10 mm in one Nitella sprigwas measured with a filar micrometer. Then the internodes were incubated in theexperimental media and the subsequent length of each internode was measured atvarious intervals. The percentage of elongation beyond the initial length was calculatedfor each internode and the mean of these values was determined for each experimentalclass. Between 6 and 10 internodes were used to determine each point. O—O,water control; • — • , 1% (v/v) dimethylsulphoxide; • — Q , 30/tg/ml of cyto-chalasin; • —• , 1/tg/ml of cycloheximide; V—V, 10 fig/ml of cycloheximide;V—V, 30 /*g/ml of cycloheximide.

Table 2. [3H]leucine uptake by Avena sections

Three-day-old, dark-grown, leafless coleoptile sections 13 mm long were incubated in2 /tCi/ml of L-[4,5-3H]leucine. Ten sections were used for each of 3 replicates of one experi-mental treatment. For preparative procedures see Methods.

Incubationtime, h

2

2 0

Experiment

ControlDimethylsulphoxide,


30 /tg/ml

ControlDimethylsulphoxide,


*(n) = number

dpm/section %(n)* change

6-48 x io4 (4) —5-83 x io4 (4) -10-5

5-34 x i o 4 (4) -18-0

5-66 x io5 (3) —6-87 XIO5 (3) +21-4

5-26 x i o 5 (3) - 7-1

of experiments performed.

Probability

> 0-05

> 0-05

> 005

> 0-05

334 M. 0. Bradley

Table 2, could lower the specific activity of the leucine pools enough to decrease incor-poration. Another possibility is that the inhibition of streaming itself prevents normalequilibration of intracellular leucine pools. And, of course, after 20 h of treatment, thedrug may become toxic.

The elongation of Nitella internodes is partially inhibited by cytochalasin (Fig. 3).This effect is slight at first, but increases to approximately 50 % inhibition after 6 hof drug treatment. As in Avena it is not yet possible to decide whether this inhibitionis a direct toxic effect of the drug itself, or a secondary effect due to the cessation ofstreaming.

Nitella ultrastructure: cytochalasin-treated

Because the general Nitella cell structure, seen here, is quite similar to that reportedby Nagai & Rebhun (1966), there is no need to describe it further. However, it isimportant to emphasize that the microfilaments are organized into widely spaced(0-7-2-0 /«n), discrete bundles that follow the streaming axis of the cell on the endo-plasmic side of the chloroplasts. Thus, the filaments must not be pictured as continuoussheets of filamentous material alongside the chloroplasts. Furthermore, the filamentbundles often appear to be closely associated with the extensive endoplasmic reticulum(Figs. 4—6, 9) and in some cases seem to end in an attachment to it (Figs. 6, 9).

In this work the individual filaments within a transversely sectioned bundle areapproximately 6-5-7-0 nm in diameter. Short projections appear to radiate from onefilament to its neighbours, so that the entire bundle seems interconnected (Fig. 7).The projections vary in diameter from 2-0 to 3-0 nm. Whether or not such projections(which might be designated as side arms, cross-bridges, cross-links, etc.) deservefunctional implications is not clear at the moment.

Fig. 4 shows that the 1 % DMSO used to dissolve cytochalasin in water, has noeffect by itself on Nitella ultrastructure. The appearance of Nitella cells after 2 h oftreatment with cytochalasin was examined in both longitudinal (Fig. 6) and transversesections (Figs. 5, 7). The drug has produced no evident ultrastructural change in eitherthe microfilaments themselves or in the other structural elements of the cell. In par-ticular, the close association of filament bundles with the endoplasmic reticulum andthe chloroplasts is maintained. There is a complete absence of the type of cytochalasin-produced masses of short filamentous material seen in drug-treated chick oviduct,mouse salivary gland, and ascidian tadpole tail.

Both the diameter of the microfilaments and the dimensions of the bundles areunchanged by cytochalasin. Also, the filament density in cross-sectioned bundles isnearly the same, with 5-7 x io~3 filaments per nm2 in control cells and 6-9 x io~3

filaments per nm2 in cytochalasin-treated cells.Cells were kept in the drug for 12 h before fixation in order to test whether the

morphological alteration in filaments seen in other systems was here a secondary effectof the drug that occurred after streaming was stopped. However, even after suchextended cytochalasin treatment, the filament ultrastructure was still maintained

(Fig- 9)-An unusual tubule complex is found in interphase nuclei (Fig. 8). The 'tubules'


are oval with a long axis of 23 nm and a short axis of 17 nm. Their length is at presentundetermined, although lengths of 600 nm have been measured. The individualtubules are packed tightly together in groups of up to 15 or 20. The tubule walls areonly 2-0-3-0 nm thick and the large, seemingly hollow core is from 11-0-17-0 nmwide (depending upon which axis of the oval is measured). Whether these tubuleaggregates are normal constituents of interphase nuclei or whether they are simplyalgal viruses cannot yet be determined. To the best of our knowledge, structures ofthis type within interphase nuclei have not been previously described in the literature.

DISCUSSION

The data presented here, in combination with previous studies (Nagai & Rebhun,1966; O'Brien & Thimann, 1966; Picket-Heaps, 1967), seem to exclude microtubulesfrom playing a major role in plant rotational streaming on several counts. First, Nitellamicrotubules are found only in the ectoplasm on the opposite side of the chloroplastsfrom the moving stream (Nagai & Rebhun, 1966; and our unpublished observations).The chloroplast files would seem to be a structural barrier, preventing the tubulesfrom acting mechanically in the endoplasm.

Secondly, although the orientation of the microtubules is not completely established,most tubules do appear to be oriented at angles oblique or perpendicular to thestreaming axis. If force is to be transmitted along the length of the tubules (as isassumed to be the case for spindle function, axoplasmic flow, etc.), then the long axisof the tubules should parallel the direction of streaming. But, in Nitella, these 2orientations are not the same.

Third, if microtubules were required to generate streaming, then colchicine shouldprevent streaming. However, streaming proceeds in both Nitella and Avena in thepresence of colchicine concentrations (Fig. 2) that are known to disrupt microtubulesin Triticum (Picket-Heaps, 1967) and Nitella (Green, 1962).

Finally, colchicine does not alter the recovery of streaming from cytochalasininhibition (Fig. 2). This finding implies that there is no interaction between micro-tubules and the cytochalasin-sensitive components of the streaming apparatus. Takingall of these data together, it can be concluded that microtubules play an insignificantrole in the types of cytoplasmic streaming that are characteristic of Nitella and Avena.

Microfilaments, 5-0-7-0 nm in diameter, have already been proposed as generatingthe motive force for streaming (see the Introduction). The fact that cytochalasin Breversibly inhibits cytoplasmic streaming is also consistent with this proposal.Previous studies have shown that in cytochalasin-treated epithelial cells of chickoviduct (Wrenn & Wessells, 1970), mouse salivary gland (Spooner & Wessells, 1970),ascidian tadpole tail (Lash, Cloney & Minor, 1970; Bradley & Wessells, in preparation),and in cleaving marine eggs (Schroeder, 1969), the respective biological phenomenon isinhibited and bundles of microfilaments, grossly similar to those in Nitella on mor-phological grounds, are significantly altered. These results imply that microfilamentsare at least one of the drug's targets of action. However, in migratory animal cells,another class of 5-nm diameter cytoplasmic filament, the sheath filament, is notaltered by cytochalasin (Spooner, Yamada & Wessells, 1971).

336 M. 0. Bradley

In Nitella, there is no evident ultrastructural change in the filament bundles, evenafter 12 h of cytochalasin treatment (Fig. 9). This means that the correlation betweenbiological effect and altered filament ultrastructure cannot be made for Nitella as it canbe for some of the other cell systems. However, because the molecular mechanism ofcytochalasin action is unknown, one does not know whether the ultrastructural effectsobserved before are necessary consequences of drug action or merely secondary effectsof the drug. Failure of cytochalasin to alter filament ultrastructure does not necessarilymean that filaments are not involved in a cytochalasin-inhibited phenomenon. Thus,a final interpretation of the fact that cytochalasin inhibits cytoplasmic streaming (aswell as other phenomena) depends upon a rigorous knowledge of the drug's mechanismof action.

Other authors have published alternative interpretations of cytochalasin's targetof action (Bluemink, 1971; Estensen, 1971; Hammer, Sheridan & Estensen, 1971).They believe that the drug inhibits processes such as membrane fusion or cell junctionformation. None of the observations of cytochalasin-disrupted filaments necessarilyexclude such interpretations, and because actomyosin-type proteins may be presentin outer cell membranes (Groschel-Stewart, Jones & Kemp 1970), it is reasonable thatthe drug acts against contractile proteins inside the cell, as well as at its surface.

Data from various animal systems (Estensen, 1971; Spooner et al. 1971; Yamada,Spooner & Wessells, 1971) indicate that cytochalasin has little effect on protein syn-thesis for at least 18 h. The data reported here suggest that plant protein synthesis andgrowth is more readily inhibited by the drug. In Avena, [3H]leucine incorporation isinhibited after 20 h in cytochalasin but not after 2 h, as shown in Table 1. Nitellainternode elongation begins to decrease within about 3 h (Fig. 3). Whether theseinhibitions are due to a direct toxic effect of cytochalasin or whether they are secondaryeffects dependent upon the prior halt in streaming, is not known. It is apparent, how-ever, that any cytochalasin inhibition of protein synthesis does not account for theeffect of the drug on streaming, since direct inhibition of protein synthesis with cyclo-heximide has no effect on streaming (Fig. 2). Furthermore, the drug's effect onstreaming takes place much more rapidly (15-45 min) than its slight inhibition ofprotein synthesis or growth.

Model for streaming

The following speculative model seeks to provide a mechanical basis for the rota-tional cytoplasmic streaming characteristic of Nitella.

In considering mechanisms of microfilament shear force generation it is pertinent topoint out that the small microfilament bundles (approximately o-i /


identical to the microfilament bundles seen at the ultrastructural level) can only movethose endoplasmic particles that are very close to them.

Of additional interest is the fact that cross-sections of Nitella (Figs. 4, 5; Nagai &Rebhun, 1966) show that an extensive endoplasmic reticulum runs as parallel planarsheets between the filaments and the inner endoplasm where streaming occurs. Itappears as if these membranes would create a structural barrier, mechanically isolatingthe filaments from the stream.

One way to solve these problems is to assume that the endoplasmic reticulum actsas a mechanical transducer, effectively coupling the filaments to the streaming endo-plasm. This idea implies that there is an interaction between the filaments and theendoplasmic reticulum such that a shear force is generated on the surfaces of thereticulum.

For instance, if sequential actomyosin cross-bridge attachments were made andbroken between the filaments and suitable sites on the endoplasmic reticulum, thenthe reticulum would move past the filaments and around the cell. In this case theviscous endoplasm would be propelled by the frictional forces transmitted to it by thelarge area of the sliding membranes. Another possibility is that a suitable mechanicalcoupling between the filament bundles and the reticulum could create travelling wavesalong the membranes that would propel the endoplasm; the principle is essentiallythe same as that for a laboratory peristaltic pump, where wave deformations along oneside of the tubing cause liquid flow. There are other mechanisms that could accountfor shear force generation on the surfaces of the reticulum; the examples presentedhere are meant to be only suggestive.

One interesting test of this mechanism would be to examine the ultrastructure ofKamitsubo's (1966a, b, 1972) centrifuged internodes and to correlate the resumptionof mass streaming with any accompanying ultrastructural changes. Will massstreaming begin as endoplasmic reticulum becomes associated with the filamentbundles?*

I wish to express my thanks to Dr Norman K. Wessells, under whom this work was done, forelectron micrographs, for advice and for enthusiastic criticism. I also thank Drs Paul Green,Peter Hepler, Peter Ray, Terry Ray, Joan Wrenn and Zac Cande for aid and helpful discussions.During the course of this work I was supported by the National Science Foundation and theState of California.

REFERENCESALLEN, R. D. & KAMIYA, N., eds. (1964). Primitive Motile Systems in Cell Biology. New York

and London: Academic Press.BIKLE, D., TILNEY, L. G. & PORTER, K. R. (1966). Microtubules and pigment migration in the

melanophores of Fundulus heteroclitus L. Protoplasma 61, 322-345.BLUEMINK, J. G. (1971). Effects of cytochalasin B on surface contractility and cell junction

formation during egg cleavage in Xenopus laevis. Cytobiologie 3, 176-187.

* While these experiments were in progress we were informed that D. G. Van Wie, then ofHarvard University, had also observed the effects of cytochalasin on cytoplasmic streaming inNitella.

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BRAY, G. (i960). A simple efficient liquid scintillator for counting aqueous solutions in a liquidscintillation counter. Analyt. Biochem. 1, 279-285.

BUCKLEY, I. K. & PORTER, K. R. (1967). Cytoplasmic fibrils in living cultured cells. Protoplasma64. 349-38o.

CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature, Lond. 213, 261-264.ESTENSEN, R. D. (1971). Cytochalasin B I: Effect on cytokinesis of Novikoff hepatoma cells.

Proc. Soc. exp. Biol. Med. 136, 1256-1260.GREEN, P. B. (1962). Mechanism for plant cellular morphogenesis. Science, N.Y. 138, 1404-

1405-GROSCHEL-STEWART, U., JONES, B. M. & KEMP, R. B. (1970). Detection of actomyosin-type

protein at the surface of dissociated embryonic chick cells. Nature, Lond. 227, 280-281.HAMMER, M. G., SHERIDAN, J. D. & ESTENSEN, R. D. (1971). Cytochalasin B I I : Selective

inhibition of cytokinesis in Xenopus laevis eggs. Proc. Soc. exp. Biol. Med. 136, 1158-1162.

HAYASHI, T . (1964). Role of the cortical gel layer in cytoplasmic streaming. In Primitive MotileSystems in Cell Biology (ed. R. D. Allen & N. Kamiya), pp. 19-29. New York and London:Academic Press.

JAROSCH, R. (1964). Screw-mechanical basis of protoplasmic movement. In Primitive MotileSystems in Cell Biology (ed. R. D. Allen & N. Kamiya), pp. 599-622. New York and London:Academic Press.

KAMITSUBO, E. (1966a). Motile protoplasmic fibrils in cells of Characeae. I. Movement offibrillar loops. Proc. Japan Acad. 42, 507-511.

KAMITSUBO, E. (19666). Motile protoplasmic fibrils in cells of Characeae. II. Linear fibrillarstructure and its bearing on protoplasmic streaming. Proc. Japan Acad. 42, 640-643.

KAMITSUBO, E. (1972). Motile protoplasmic fibrils in cells of Characeae. Protoplasma 74, 53-70.KAMIYA, N. (1959). Protoplasmic streaming. Protoplasmatologia 8, 3 a, 167-169.KAMIYA, N. (i960). Physics and chemistry of protoplasmic streaming. A. Rev. PL Physiol. 11,

323-340.KAMIYA, N. & KURODA, K. (1956). Velocity distribution of the protoplasmic streaming in

Nitella cells. Bot. Mag., Tokyo 69, 544-554.KAMIYA, N. & KURODA, K. (1963). Rotational protoplasmic streaming in Nitella and some

physical properties of the endoplasm. In Proc. \th int. Congr. Rheology, part 4: Symposiumon Biorheology, pp. 157-168. New York: John Wiley & Sons.

KREUTZBERG, G. W. (1969). Neuronal dynamics in axonal flow. IV. Blockage of intra-axonalenzyme transport by colchicine. Proc. natn. Acad. Sci. U.S.A. 62, 722-728.

LASH, J., CLONEY, R. A. & MINOR, R. R. (1970). Tail resorption in ascidians: Effects of cyto-chalasin B. Biol. Bull. mar. biol. Lab., Woods Hole 139, 427-428.

LEDBETTER, M. C. & PORTER, K. R. (1963). A ' microtubule' in plant cell fine structure. J. CellBiol. 19, 239-250.

LEDBETTER, M. C. & PORTER, K. R. (1964). Morphology of microtubules of plant cells. Science,N. Y. 144, 872-874.

LOWRY, H. O., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, D. J. (1951). Protein measurementwith the Folin phenol reagent. J. biol. Chem. 193, 265-275.

MCINTOSH, J. R., HEPLER, P. K. & VAN WIE, D. G. (1969). Model for mitosis. Nature, Lond.224, 659-663.

NAGAI, R. & REBHUN, L. I. (1966). Cytoplasmic microfilaments in streaming Nitella cells.J. Ultrastruct. Res. 14, 571-589.

O'BRIEN, T . P. & THIMANN, K. V. (1966). Intracellular fibers in oat coleoptile cells andtheir possible significance in cytoplasmic streaming. Proc. natn. Acad. Sci. U.S.A. 56,888-894.

PARTHASARATHY, M. V. & MUHLETHALER, K. (1972). Cytoplasmic microfilaments in plantcells. J. Ultrastruct. Res. 38, 46-62.

PICKETT-HEAPS, J. D. (1967). The effects of colchicine on the ultrastructure of dividing plantcells, xylem wall differentiation and distribution of cytoplasmic microtubules. Devi Biol. 15,206-236.

POLLARD, T . D. & ITO, S. (1970). Cytoplasmic filaments of Amoeba proteus. I. The role offilaments in consistency changes and movement. J. Cell Biol. 46, 267-289.


REBHUN, L. I. (1967). Structural aspects of saltatory particle movement, jf. gen. Physiol. 50,Suppl., 223-239.

SABNIS, D. D. & JACOBS, W. P. (1967). Cytoplasmic streaming and microtubules in the coeno-cytic marine alga, Caulerpa prolifera. J. Cell Sci. 2, 465-471.

SCHROEDER, T. E. (1969). The role of 'contractile ring' filaments in dividing Arbacia egg. Biol.Bull. mar. biol. Lab., Woods Hole 137, 413-414.

SPOONER, B. S. & WESSELLS, N. K. (1970). Effects of cytochalasin B upon microfilamentsinvolved in morphogenesis of salivary epithelium. Proc. natn. Acad. Sci. U.S.A. 66, 360-364.

SPOONER, B. S., YAMADA, K. & WESSELLS, N. K. (1971). Microfilaments and cell locomotion.jf. Cell Biol. 49, 595-613.

TILNEY, L. G. (1968). Studies on the microtubules in Heliozoa. IV. The effect of colchicine onthe formation and maintenance of the axopodia and the redevelopment of pattern in Actino-sphaerium nucleofilum (Barrett), jf. Cell Sci. 3, 549-562.

TILNEY, L. G. & PORTER, K. R. (1965). Studies on microtubules in Heliozoa. I. The fine struc-ture of Actinosphaerium nucleofilum (Barrett) with particular reference to the axial rodstructure. Protoplasma 60, 317-344.

VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electronmicroscopy, jf. Cell Biol. z$, 407-408.

WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR,

E. L., WRENN, J. T . & YAMADA, K. M. (1971). Microfilaments in cellular and developmentalprocesses. Science, N.Y. 171, 135-143.

WOHLFARTH-BOTTERMANN, K. E. (1964). Differentiations of the ground cytoplasm and theirsignificance for the generation of motive force of ameboid movement. In Primitive MotileSystems in Cell Biology (ed. R. D. Allen & N. Kamiya), pp. 79-109. New York and London:Academic Press.

WOHLMAN, A. & ALLEN, R. D. (1968). Structural organization associated with pseudopodextension and contraction during cell locomotion in Difflugia. J. Cell Sci. 3, 105-114.

WRENN, J. T. & WESSELLS, N. K. (1970). Cytochalasin B: Effects upon microfilaments involvedin morphogenesis of estrogen-induced glands of oviduct. Proc. natn. Acad. Sci. U.S.A. 66,904-908.

YAMADA, K. M., SPOONER, B. S. & WESSELLS, N. K. (1971). Ultrastructure and function ofgrowth cones and axons of cultured nerve cells, jf. Cell Biol. 49, 614-635.

(Received 17 March 1972)

340 M.O. Bradley

All micrographs are of Nitella internodes, fixed with glutaraldehyde and osmiumtetroxide.

Fig. 4. Transverse section of an internode treated with 1 % DMSO for 2 h. The ultra-structure has not been affected by the DMSO. The streaming axis is normal to theplane of the micrograph. A bundle of microfilaments (mf) is present between thechloroplast (c) and the rich endoplasmic reticulum (er). This micrograph shows mainlythe endoplasm interior to the chloroplasts; the ectoplasm extends above the chloro-plasts. x 22 500.

Fig. 5. Transverse section showing a microfilament bundle (mf) just under thechloroplast (c) and in close proximity to the endoplasmic reticulum (er). This internodewas treated with 30 fig/ml of cytochalasin for 2 h. x 115 000.

Fig. 6. Longitudinal section showing a band of microfilaments (mf) that approachesclosely to the endoplasmic reticulum (er). The filaments extend beyond one chloro-plast (c) in the direction of the next. This internode was treated with 30 /


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342 M. O. Bradley

Fig. 7. Transverse section showing presumed microfilament 'side-arms' (sa). Shortprojections seem to interconnect one filament with the others surrounding it. Thiscell was incubated in 30 /tg/ml of cytochalasin for 2 h before fixation. 'Side-arms' areseen in untreated filaments as well, x 195000.

Fig. 8. Transverse section of an interphase nucleus showing presumptive 'nucleartubules' (nt). x 150000.

Fig. 9. Longitudinal section of an internode treated with 30 /tg/ml of cytochalasin for12 h. The general cell structure is unchanged and the microfilament bundles (mf)still approach the endoplasmic reticulum (er). x 130000.

Ejfect of cytochalasin on streaming 343

microfilaments and cytoplasmic streaming: inhibition of streaming … · 2005. 9. 6. · 33° m. 0....

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