Microfilaments and Cytoplasmic Streaming: Inhibition of Streaming with Cytochalasin

J. Cell Sci. 12, 327-343 (i973) 327 Printed in Great Britain 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has no effect on streaming in either plant; nor does colchicine prevent the recovery of streaming after cytochalasin is withdrawn.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. Micro- tubules, 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. Micro- filament-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 (Wohlfarth- Bottermann, 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. Micro- tubules, 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 sterile filtered pond 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 005 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 rever- sal 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 71. They were then washed 5 times over a period of 30mm with 0025 M potassium phosphate buffer, pH7-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 71 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 incuba- tion the sections were chilled, washed 4 times with unlabelled leucine at io4 times the concentration of the labelled leucine (io4 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 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) with io4 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 io4xcold leucine. Protein was digested from the dried filters with 12 ml of NCS. The digests, 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 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.

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