J. Cell Sci. 12, 327-343 (i973)
Printed in Great Britain
327
MICROFILAMENTS AND CYTOPLASMIC
STREAMING: INHIBITION OF STREAMING
WITH CYTOCHALASIN
M. O. BRADLEY
Department 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 colchicine
prevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of protein
synthesis by cycloheximide has no effect on either streaming itself or on the recovery of
streaming after cytochalasin withdrawal. All this suggests that microfilaments may provide one
component of the structure that generates the streaming force and that microtubules play
little, if any, role in the process.
Ultrastructural studies of Nitella demonstrate that microfilaments are localized at the
boundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments are
organized in discrete bundles, with possible cross-bridges between individual filaments in each
bundle. These bundles are closely associated with the extensive endoplasmic reticulum.
Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does in
some 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 altered
microfilament morphology.
A model is advanced which proposes that streaming is generated by an interaction between
microfilaments and the endoplasmic reticulum.
INTRODUCTION
Cytoplasmic streaming occurs in a great variety of plant and animal cells (see
Kamiya, i960; Allen & Kamiya, 1964, for reviews). The forces that direct such
streaming are not well understood; however, since cytoplasmic particles do not move
by the action of forces generated by the particles themselves, some external cytoplasmic
forces must be moving them (Rebhun, 1967). The fibrillar microtubule and microfilament systems are possible candidates for this force-generating role and have been
hypothesized as the structural elements driving various types of streaming. Microtubules, for instance, have been implicated in 'fast' axoplasmic transport in nerve
cells (Kreutzberg, 1969), in the streaming and saltatory movements of heliozoan
axopods (Tilney & Porter, 1965; Tilney, 1968), in melanin granule migration in fish
melanophores (Bikle, Tilney & Porter, 1966), in chromosome movement in the mitotic
apparatus (Mclntosh, Hepler & Van Wie, 1969), and in higher plant (Ledbetter &
Porter, 1963, 1964) and algal (Sabnis & Jacobs, 1967) cytoplasmic streaming. Microfilament-based motility has been proposed for phenomena such as cytoplasmic
*Address for reprint requests: Department of Medical Microbiology, Stanford University
School of Medicine, Stanford, California, 94305, U.S.A.
328
M. 0. Bradley
streaming and movement of Amoeba (Pollard & Ito, 1970), Physarum (WohlfarthBottermann, 1964), and Difflugia (Wohlman & Allen, 1968), organelle movement in
cultured rat embryo cells (Buckley & Porter, 1967), glia, nerve and fibroblast movement 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 these
phenomena is based on a correlation between the observed biological event and the
spatial localization of the respective organelle. In some cases both microtubules and
microfilaments are found in the same locus and so permit alternative explanations for
a given phenomenon.
In Nitella, Kamiya & Kuroda (1956, 1963) and Hayashi (1964) have shown by
physical measurements that the motive force for streaming is localized at the interface
separating the flowing endoplasm from the stationary cortical gel layer. At this interface, Nagai & Rebhun (1966) found large bundles of 5-nm diameter microfilaments
oriented with their long axis parallel to the direction of streaming. They proposed that
these microfilament bundles generate the motive force for rotational streaming. Microtubules, on the other hand, are located just below the plasma membrane in the
stationary ectoplasm. Since they are on the opposite side of the stationary chloroplasts
from the streaming endoplasm, and since they are not necessarily oriented parallel to
the axis of streaming, microtubules are considered less likely to be of importance in the
streaming phenomenon.
The site at which the motive force is generated has not been determined by physical
techniques in Avena, as it has been for Nitella. Nevertheless, O'Brien & Thimann
(1966) found bundles of 5-nm microfilaments in Avena epidermal and parenchymal
cells, and hypothesized that Avena streaming also depends upon such filaments. As
in Nitella, the Avena filament bundles parallel the direction of streaming.
Drugs that selectively attack these filamentous organelles can be used to test the
validity of different hypotheses and to discriminate between microtubule and microfilament based processes. This paper pursues this approach in an investigation of cytoplasmic streaming in the alga Nitella and the higher plant Avena by utilizing cytochalasin B (Carter, 1967; Wessells et al. 1971) and colchicine (Pickett-Heaps, 1967),
drugs that are thought to attack microfilaments and microtubules respectively. A
preliminary 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 cytoplasmic
streaming.
MATERIALS AND METHODS
Plants
Experiments 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 sterilefilteredpond
water or in glass-distilled water. All experimental dishes were kept on a light tray except during
observations.
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 oxygenated
0 0 5 M sodium phosphate buffer, pH 7-5, with 1-5% (w/v) sucrose added. Control streaming
continued for from 10 to 15 h under these conditions.
Streaming measurements
For Nitella, the rates of endoplasmic particle movement were measured with a stopwatch
and an ocular micrometer.
For Avena, rates of particle movement were not measured because of great intracellular
variations in rate. Instead, qualitative effects of different drug treatments were assayed by noting
whether streaming in a section was vigorous in all cells, slowing or stopped in some cells, or
completely 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 in
dimethylsulphoxide (DMSO) and diluted to the appropriate concentration with medium. The
final DMSO concentration in experimental and control cultures was always 1 % (v/v). In reversal experiments, cytochalasin was removed by washing the plants with 5 changes of drug-free
medium.
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 added
to the media in order to inhibit protein synthesis before and after recovery from cytochalasin
treatment.
Electron microscopy
Nitella internodes, 1 to 2 cm long, were fixed for 15 h at room temperature with a solution
of 3 % glutaraldehyde in 0006 M potassium phosphate buffer pH 7 1 . They were then washed
5 times over a period of 3 0 m m with 0025 M potassium phosphate buffer, p H 7 - i . After
washing, 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. The
sections were washed again with phosphate buffer and then dehydrated through an ethanol
series (15 to 100%) overnight. Following clearing with propylene oxide, embedding was done
in Epon. Thin sections were cut on a Sorvall MT-2 ultramicrotome, stained with uranyl acetate
and lead citrate (Venable & Coggeshall, 1965) and examined with an Hitachi HU-11E electron
microscope.
Radioisotope techniques
Uptake. Avena seedlings were grown for 3 days in the dark. Leafless coleoptile sections 13 mm
long were incubated for varying lengths of time in 2 /tCi/ml of L-[4,5-3H]leucine. After incubation the sections were chilled, washed 4 times with unlabelled leucine at io 4 times the concentration of the labelled leucine (io 4 x leucine), chopped into short sections and washed again 4
times. 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 the
samples were counted with a Nuclear Chicago Mark II. Disintegrations per min (dpm) were
calculated by the channels-ratio method.
Incorporation. The Avena sections were treated in the same way as for the uptake studies until
after the final washing. Then the sections were sonicated for 2 min with a Branson sonicator
in io 4 x leucine. The method of Lowry, Rosebrough, Farr & Randall (1951) was used to determine the protein concentration of the sonicate. Cold, 1 0 % trichloroacetic acid (TCA) with
io 4 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 % TCA
plus io 4 xcold leucine. Protein was digested from the dried filters with 1 2 ml of NCS. The
digests, including the filters, were counted using the procedures outlined above.
M. 0. Bradley
33°
Fig. i. The effect of cytochalasin on cytoplasmic streaming in Nitella internode cells
Data from 3 typical cells are shown. At the times indicated, 30 /*g/ml of cytochalasin was
added ( + cb) and later removed ( - cb) by washing the cells with 5 changes of cytochalasin-free medium. O—O, control; A—A, 1 % (v/v) dimethylsulphoxide; % %
30 /tg/rnl cytochalasin.
RESULTS
Effects 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 drug
than the Nitella internodes. Thus, Nitella is insensitive at concentrations of 3 /tg/ml
or less, while Avena is insensitive below 1 /tg/ml. Avena streaming was stopped within
30 min to 1 h by 30 /tg/ml of cytochalasin.
Just before streaming is stopped, cytoplasmic particles in both systems move in
parallel short jerks, often at angles oblique to the original streaming axis. The Nitella
stream 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 though
they continue to move forward at approximately 5 /tm/s.
If the plants are washed with cytochalasin-free medium, streaming begins again
within 15-30 min in both plants. In Nitella, the 'recovered' streaming appears normal
and 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. Nitella
recovery will occur after as long as 12 h of arrested streaming due to continuous drug
treatment. Although shorter term treatment appears to be harmless, after 24 h in cytochalasin Nitella cells begin to degenerate and by 36 h most are dead. This is indicated
by the clumping of the cytoplasm and the randomization of the chloroplast files. Such
long term cytotoxity could be the result of the drug itself, or it could be a secondary
effect due to the cessation of streaming.
Effect of cytochalasin on streaming
331
+ cb
Fig. 2. The effect of combinations of cytochalasin, colchicine, and cycloheximide on
cytoplasmic streaming in Nitella. Data from 4 typical internodes are shown. At the
times 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 and
removed at the indicated times; V —V, 1 o /*g/ml of cycloheximide; V —T, 10 /tg/ml
cycloheximide with 30 /tg/ml of cytochalasin added and removed at the indicated
times.
Effects of colchicine on streaming
Colchicine was applied to Nitella and Avena cells in order to study the role of microtubules in cytoplasmic streaming. Colchicine has no effect upon streaming in Nitella
for at least 24 h (Fig. 2), or in Avena for 10 h.
Possible interactions between microtubules and microfilaments were examined by
permitting cells to 'recover' from cytochalasin treatment in the presence of colchicine.
In this experiment Avena sections and Nitella internodes were preincubated for 2 h in
colchicine. Then 30/tg/ml of cytochalasin was added until streaming stopped; 1 h
later cytochalasin was washed out and the plants were allowed to 'recover' in the
presence of colchicine alone. Both Nitella (Fig. 2) and Avena streaming 'recover'
with the same kinetics as in colchicine-free controls. These findings all suggest that
microtubules play an insignificant role in streaming generation.
Effects of cycloheximide on streaming
Cycloheximide, an inhibitor of protein synthesis occurring on polysome-bound
80-s ribosomes, was applied to normally streaming plants and to those 'recovering'
from cytochalasin. Both Nitella (Fig. 2) and Avena streaming is unaffected by longterm cycloheximide treatment. Furthermore, the drug allows normal 'recovery' from
cytochalasin inhibition of streaming. These results suggest that the proteins of the
streaming 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]leucine
incorporation by Avena coleoptiles
Three-day-old, darkgrown, leafless coleoptile sections 13 mm long were incubated for 2 or
20 h in 2 fiCi/ml of L-[4,5-3H]leucine. Ten sections were used for each of 3 replicates of one
experimental treatment. For preparative procedures see Methods.
Labelling
time, h
2
2O
Experiment
dpm/mg
protein (n)*
0/
/o
change
Probability
4
Control
Cycloheximide,
665 x io (3)
9 1 0 X i o 3 (3)
-863
<
0001
Dimethylsulphoxide,
1 % v/v
Cytochalasin,
30 /*g/ml
Control
Cycloheximide,
io/^g/ml
Dimethylsulphoxide,
1 % v/v
Cytochalasin,
30 //g/ml
6-31 x io 4 (3)
-5'4°
>
005
6-50 x io 4 (3)
— 260
>
005
6-68 x io 6 (3)
1 09 x io 5 (3)
-836
<
0001
5-87 x i o 5 (3)
— I2-O
>
005
3-40 x 1 o5 (3)
-42-2
<
0001
—
* (n) = number of experiments performed
tional streaming apparatus after cytochalasin inhibition requires neither immediately
prior nor concomitant protein synthesis.
The dose of cycloheximide used in these experiments decreased [3H]leucine incorporation into Avena coleoptiles by 86-3 % in 2 h and by 83-6 % in 20 h as seen in Table
1. Because an abundant bacterial microflora adheres to Nitella cell walls, it is impossible
to measure protein synthesis directly in this system. Instead, we determined the effect
of cycloheximide on the growth rate of Nitella internodes, making the assumption that
growth rate and protein synthesis must be coupled in some way. The results show
that cycloheximide completely stops internode elongation (Fig. 3); this is consistent
with 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 incorporation into presumed protein in Avena sections. During a 2-h labelling period, there
is no significant decrease in the number of counts incorporated. However, after 20 h
of continuous labelling, total incorporation is decreased by 42 %. Because cytochalasin
is applied for only 2 or 3 h in most experiments dealing with effects on streaming, and
because direct inhibition of protein synthesis with cycloheximide has no effect on
streaming, it is clear that the rapid biological effects of the drug cannot be explained
by decreased rates of protein synthesis.
The appreciable decrease in long term incorporation is not yet understood. One
explanation is that the partial cytochalasin inhibition of leucine uptake (if real), seen in
Effect of cytochalasin on streaming
333
25 -
Fig. 3. The effect of cycloheximide and cytochalasin on elongation of Nitella internodes. The initial length of every internode between 2 and 10 mm in one Nitella sprig
was measured with a filar micrometer. Then the internodes were incubated in the
experimental media and the subsequent length of each internode was measured at
various intervals. The percentage of elongation beyond the initial length was calculated
for each internode and the mean of these values was determined for each experimental
class. Between 6 and 10 internodes were used to determine each point. O—O,
water control; • — • , 1% (v/v) dimethylsulphoxide; • — Q , 30/tg/ml of cytochalasin; • — • , 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 in
2 /tCi/ml of L-[4,5-3H]leucine. Ten sections were used for each of 3 replicates of one experimental treatment. For preparative procedures see Methods.
Incubation
time, h
2
Experiment
Control
Dimethylsulphoxide,
dpm/section
(n)*
%
change
6-48 x io4 (4)
—
5-83 x io 4 (4)
-10-5
> 0-05
5-34 x i o 4 (4)
-18-0
> 0-05
Probability
1 % v/v
Cytochalasin,
30 /tg/ml
20
Control
Dimethylsulphoxide,
5-66 x io 5 (3)
—
6-87 XIO5 (3)
+21-4
> 005
5-26 x i o 5 (3)
-
> 0-05
1 % v/v
Cytochalasin,
*(n) = number of experiments performed.
7-1
334
M. 0. Bradley
Table 2, could lower the specific activity of the leucine pools enough to decrease incorporation. Another possibility is that the inhibition of streaming itself prevents normal
equilibration of intracellular leucine pools. And, of course, after 20 h of treatment, the
drug 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 h
of drug treatment. As in Avena it is not yet possible to decide whether this inhibition
is a direct toxic effect of the drug itself, or a secondary effect due to the cessation of
streaming.
Nitella ultrastructure: cytochalasin-treated
Because the general Nitella cell structure, seen here, is quite similar to that reported
by Nagai & Rebhun (1966), there is no need to describe it further. However, it is
important 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 endoplasmic side of the chloroplasts. Thus, the filaments must not be pictured as continuous
sheets of filamentous material alongside the chloroplasts. Furthermore, the filament
bundles 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 are
approximately 6-5-7-0 nm in diameter. Short projections appear to radiate from one
filament 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.) deserve
functional implications is not clear at the moment.
Fig. 4 shows that the 1 % DMSO used to dissolve cytochalasin in water, has no
effect by itself on Nitella ultrastructure. The appearance of Nitella cells after 2 h of
treatment with cytochalasin was examined in both longitudinal (Fig. 6) and transverse
sections (Figs. 5, 7). The drug has produced no evident ultrastructural change in either
the microfilaments themselves or in the other structural elements of the cell. In particular, the close association of filament bundles with the endoplasmic reticulum and
the chloroplasts is maintained. There is a complete absence of the type of cytochalasinproduced 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 are
unchanged by cytochalasin. Also, the filament density in cross-sectioned bundles is
nearly 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 effect
of the drug that occurred after streaming was stopped. However, even after such
extended cytochalasin treatment, the filament ultrastructure was still maintained
( Fi g- 9)An unusual tubule complex is found in interphase nuclei (Fig. 8). The 'tubules'
Effect of cytochalasin on streaming
335
are oval with a long axis of 23 nm and a short axis of 17 nm. Their length is at present
undetermined, although lengths of 600 nm have been measured. The individual
tubules are packed tightly together in groups of up to 15 or 20. The tubule walls are
only 2-0-3-0 nm thick and the large, seemingly hollow core is from 11-0-17-0 nm
wide (depending upon which axis of the oval is measured). Whether these tubule
aggregates are normal constituents of interphase nuclei or whether they are simply
algal viruses cannot yet be determined. To the best of our knowledge, structures of
this 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 microtubules
from playing a major role in plant rotational streaming on several counts. First, Nitella
microtubules are found only in the ectoplasm on the opposite side of the chloroplasts
from the moving stream (Nagai & Rebhun, 1966; and our unpublished observations).
The chloroplast files would seem to be a structural barrier, preventing the tubules
from 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 the
streaming axis. If force is to be transmitted along the length of the tubules (as is
assumed to be the case for spindle function, axoplasmic flow, etc.), then the long axis
of the tubules should parallel the direction of streaming. But, in Nitella, these 2
orientations are not the same.
Third, if microtubules were required to generate streaming, then colchicine should
prevent streaming. However, streaming proceeds in both Nitella and Avena in the
presence of colchicine concentrations (Fig. 2) that are known to disrupt microtubules
in Triticum (Picket-Heaps, 1967) and Nitella (Green, 1962).
Finally, colchicine does not alter the recovery of streaming from cytochalasin
inhibition (Fig. 2). This finding implies that there is no interaction between microtubules and the cytochalasin-sensitive components of the streaming apparatus. Taking
all of these data together, it can be concluded that microtubules play an insignificant
role 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 generating
the motive force for streaming (see the Introduction). The fact that cytochalasin B
reversibly inhibits cytoplasmic streaming is also consistent with this proposal.
Previous studies have shown that in cytochalasin-treated epithelial cells of chick
oviduct (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 is
inhibited and bundles of microfilaments, grossly similar to those in Nitella on morphological grounds, are significantly altered. These results imply that microfilaments
are 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 not
altered by cytochalasin (Spooner, Yamada & Wessells, 1971).
336
M. 0. Bradley
In Nitella, there is no evident ultrastructural change in the filament bundles, even
after 12 h of cytochalasin treatment (Fig. 9). This means that the correlation between
biological effect and altered filament ultrastructure cannot be made for Nitella as it can
be for some of the other cell systems. However, because the molecular mechanism of
cytochalasin action is unknown, one does not know whether the ultrastructural effects
observed before are necessary consequences of drug action or merely secondary effects
of the drug. Failure of cytochalasin to alter filament ultrastructure does not necessarily
mean that filaments are not involved in a cytochalasin-inhibited phenomenon. Thus,
a final interpretation of the fact that cytochalasin inhibits cytoplasmic streaming (as
well as other phenomena) depends upon a rigorous knowledge of the drug's mechanism
of action.
Other authors have published alternative interpretations of cytochalasin's target
of action (Bluemink, 1971; Estensen, 1971; Hammer, Sheridan & Estensen, 1971).
They believe that the drug inhibits processes such as membrane fusion or cell junction
formation. None of the observations of cytochalasin-disrupted filaments necessarily
exclude such interpretations, and because actomyosin-type proteins may be present
in outer cell membranes (Groschel-Stewart, Jones & Kemp 1970), it is reasonable that
the 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 synthesis for at least 18 h. The data reported here suggest that plant protein synthesis and
growth is more readily inhibited by the drug. In Avena, [3H]leucine incorporation is
inhibited after 20 h in cytochalasin but not after 2 h, as shown in Table 1. Nitella
internode elongation begins to decrease within about 3 h (Fig. 3). Whether these
inhibitions are due to a direct toxic effect of cytochalasin or whether they are secondary
effects dependent upon the prior halt in streaming, is not known. It is apparent, however, that any cytochalasin inhibition of protein synthesis does not account for the
effect of the drug on streaming, since direct inhibition of protein synthesis with cycloheximide has no effect on streaming (Fig. 2). Furthermore, the drug's effect on
streaming takes place much more rapidly (15-45 min) than its slight inhibition of
protein synthesis or growth.
Model for streaming
The following speculative model seeks to provide a mechanical basis for the rotational cytoplasmic streaming characteristic of Nitella.
In considering mechanisms of microfilament shear force generation it is pertinent to
point out that the small microfilament bundles (approximately o-i /<m in diameter) are
spaced at large intervals (of about 1 /im) alongside the choroplast and have a very
small area in relation to the large volume of the endoplasm. Such small area: volume
ratios imply that undulating (Kamiya, 1959) or rotating (Jarosch, 1964) filament
bundles would be unlikely to create enough shear force by themselves to drive a large
volume of even a very viscous endoplasm. Kamitsubo's (1966a, b, 1972) light-microscopic observations of motile and stationary protoplasmic fibrils in centrifuged Nitella
internodes tend to support this idea. He reports that the fibrils (which are presumably
Effect of cytochalasin on streaming
337
identical to the microfilament bundles seen at the ultrastructural level) can only move
those 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 planar
sheets between the filaments and the inner endoplasm where streaming occurs. It
appears as if these membranes would create a structural barrier, mechanically isolating
the filaments from the stream.
One way to solve these problems is to assume that the endoplasmic reticulum acts
as a mechanical transducer, effectively coupling the filaments to the streaming endoplasm. This idea implies that there is an interaction between the filaments and the
endoplasmic reticulum such that a shear force is generated on the surfaces of the
reticulum.
For instance, if sequential actomyosin cross-bridge attachments were made and
broken between the filaments and suitable sites on the endoplasmic reticulum, then
the reticulum would move past the filaments and around the cell. In this case the
viscous endoplasm would be propelled by the frictional forces transmitted to it by the
large area of the sliding membranes. Another possibility is that a suitable mechanical
coupling between the filament bundles and the reticulum could create travelling waves
along the membranes that would propel the endoplasm; the principle is essentially
the same as that for a laboratory peristaltic pump, where wave deformations along one
side of the tubing cause liquid flow. There are other mechanisms that could account
for shear force generation on the surfaces of the reticulum; the examples presented
here are meant to be only suggestive.
One interesting test of this mechanism would be to examine the ultrastructure of
Kamitsubo's (1966a, b, 1972) centrifuged internodes and to correlate the resumption
of mass streaming with any accompanying ultrastructural changes. Will mass
streaming begin as endoplasmic reticulum becomes associated with the filament
bundles?*
I wish to express my thanks to Dr Norman K. Wessells, under whom this work was done, for
electron 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 the
State of California.
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* While these experiments were in progress we were informed that D. G. Van Wie, then of
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All micrographs are of Nitella internodes, fixed with glutaraldehyde and osmium
tetroxide.
Fig. 4. Transverse section of an internode treated with 1 % D M S O for 2 h. The ultrastructure has not been affected by the DMSO. The streaming axis is normal to the
plane of the micrograph. A bundle of microfilaments (mf) is present between the
chloroplast (c) and the rich endoplasmic reticulum (er). This micrograph shows mainly
the endoplasm interior to the chloroplasts; the ectoplasm extends above the chloroplasts. x 22 500.
Fig. 5. Transverse section showing a microfilament bundle (mf) just under the
chloroplast (c) and in close proximity to the endoplasmic reticulum (er). This internode
was treated with 30 fig/ml of cytochalasin for 2 h. x 115 000.
Fig. 6. Longitudinal section showing a band of microfilaments (mf) that approaches
closely to the endoplasmic reticulum (er). The filaments extend beyond one chloroplast (c) in the direction of the next. This internode was treated with 30 /<g/ml of
cytochalasin for 2 h. x 58900.
Effect of cytochalasin on streaming
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M. O. Bradley
Fig. 7. Transverse section showing presumed microfilament 'side-arms' (sa). Short
projections seem to interconnect one filament with the others surrounding it. This
cell was incubated in 30 /tg/ml of cytochalasin for 2 h before fixation. 'Side-arms' are
seen in untreated filaments as well, x 195000.
Fig. 8. Transverse section of an interphase nucleus showing presumptive 'nuclear
tubules' (nt). x 150000.
Fig. 9. Longitudinal section of an internode treated with 30 /tg/ml of cytochalasin for
12 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