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CELL STRUCTURE AND FUNCTION 5, 13-20 (1980) by Japan Society for Cell Biology C Motile Apparatus in Vallisneria Leaf Cells. II. Effects of Cytochalasin B and Lead Acetate and Direction of Streaming on the Rate Mitsuo Ishigami* and Reiko Nagai** *Department of Biology , Faculty of Education, Shiga University, Otsu, Shiga 520, and **Department of Biology, Faculgy of Science, Osaka University, Toyonaka, Osaka 560, Japan ABSTRACT. The "primary streaming" of streaming" in streaming in of the of cytochalasin in Vallisneria and the Cytoplasmic range effects streaming 10-100 ƒÊg/ml. reverse direction observed before duration of of its was The cells and lead acetate, inhibitors examined on "secondary were with reference reversibly of inhibited cells removal increased reaching of CB depending 50 % by showing after CB newly to not apparatus induce in a change secondary in the the cessation on 24 h total the for streaming streaming are direction. discussed on in the of streaming number of concentration CB Properties cells and treatment cessation however, this concentration established the µg/ml. Lead acetate at 50-100 mM caused the complete but the streaming gradually recovered after its removal; did to direction. ratio after treatment treatment, cells, mesophyll reversal B (CB) Characean at the 10-100 of streaming, lead acetate of the motile basis. In leaf cells of the higher aquatic plants e.g., Elodea and Vallisneria, rotational streaming of the cytoplasm is induced by such external stimuli as irradiation with light or the application of various chemicals. This type of streaming is called "secondary streaming" (4, 6), and that seen in Characean cells is called "primary streaming", as it is steady and ceaseless under natural conditions. Considerable information of the mechanism of primary streaming has been obtained for the internodal cells of Characeae. The rotational cytoplasmic streaming in the Characean cells generally is understood to be caused by the unidirectional sliding of the endoplasmic organelles equipped with myosin-like protein along bundles of F-actin filaments, all of which are anchored on the stationary chloroplast files. The direction of the streaming is thought to be determined by the polarity of the F-actin composing the bundles of microfilaments (7). Less is known, however, about the structure and function of the motile system for secondary streaming, though the nature of the photoreceptor involved in light-induced cytoplasmic streaming and chloroplast movement has been clarified to some extent (3, 12, 13). Information obtained from investigations of Characean cells should help us to understand the mechanism of rotational streaming in the leaf cells of aquatic plants. Forde and Steer (2) looked for microfilament bundles in leaf cells of Elodea, but failed to find large bundles that might be responsible for the generation of cytoplasmic streaming. We recently have found many bundles of microfilaments in 13 14 M. Ishigami and R. Nagai Vallisneria leaf cells which manifested active cytoplasmic streaming (unpublished results). These bundles, which appear very similar to those in Characean cells, are localized in the peripheral region of the cell. Their orientation coincides well with the direction of streaming. Whether these bundles are generated prior to the induction and establishment of streaming attracted our interest. If so, the mechanism of secondary streaming must be closely related to the stability of these microfilament bundles. The present study was performed to obtain information on the properties of the motile apparatus in secondary streaming. Using two drugs, cytochalasin B (CB) and lead acetate, we examined i) the cessation of streaming induced by their application and ii) the reversal of the streaming direction after their removal. As stated above, the endoplasm in Characean cells streams unidirectionally, supposedly due to the polarity of the microfilaments. Reversal of the streaming direction, therefore, can occur only through the disorganization and reconstruction of the microfilament bundles. The stability of these bundles in Vallisneria in relation to the mechanism of secodary streaming is discussed. MATERIALS Cultures. Vallisneria cultured The in tap bottom Leaves layers. first slip five cells, each were was the drug on and The were streaming recorded for had been direction were the per of same cells. changed by one by The the ratio drug the water. equipped between rates, condition. The passed the rate im- 10-20 ƒÊm/sec. drug with the of number total The five and directions of to in tap inspected the part. mesophyll that of were epidermal inner streaming of the drug, river. streaming test the a range the irrigation the of 30ƒÊ a razor. IMT) particles sample the a slide measure application cells and on value of epidermal under within layers was at with the applied the usually before removal the two cytoplasmic to from region (Olympus To Japan, lux occupying of examined of twenty the cells, mounted stage. were was collected cells and Otsu, 2,000 basal microscope solutions checked in about After off normalized which was of in about sand the consisting microscope were cells directions layers cut movement application, apparatus. then of sheet, the River of at mesophyll a sheet control the piece timing in the to make off of were and Seta muddy four a phase-contrast glass divisions of layers with Test of them sheet a different streaming the of a razor 10 cm up other the illumination cutting is made two with by drawing established which from leaf. the leaf slide of streaming stripped with the before direction by apparatus. and eyepiece mediately with needed observed measured calibrated of were a drawing cover covered and from continuous was side METHODS biwaensis, when pieces cells the under stripped Small Mesophyll var. a pail The exterior were rates pail harvested each Leaves with the asiatica in Microscopy. on cell of were Light cells water AND pieces recorded the of newly cells number of in cells calculated. Chemicals. in 1, 5, 10, Lead of fore Cytochalasin dimethyl sulfoxide 50, acetate 10, 50, 100 and (Nakarai 100, application. 200 B (Imperial (DMSO), 200 ƒÊg/ml. Chemicals, and 500 mM. then A 1% Ltd.) The Chemical diluted DMSO was solution Industries) with tap water solution dissolved of lead to was in tap acetate was dissolved give final used water was for the to give prepared at 20 ƒÊg/ml concentrations control final of medium. concentrations immediately be- Motile Apparatus in Vallisneria 15 Cells RESULTS Effects of cytochalasin centration of 1, 5, concentration. For illuminated off the the B. 10, rates. irrigation. 50, about cells 30 on the for streaming to the drug Five rate the of the stage (about was at examined drug each rates, 5,500 lux) was replaced a conat streaming the solution applied were monitoring, test was cells monitoring of min, The 1 h. microscope start 60 rate: for prior light after treatment the 200 ƒÊg/m1 min the min On or 15 with Then After 1) 100 we to level applied by the by control solution. Streaming g/ml. was Fig. drug application, tion and the control 10 and was the at 5 ƒÊg/ml Fig. 1), 1. CB control, was as cells. of applied cells The CB at treated by irrigating application, 1.00; not were removed before recover other length which data were of 50 of the min. 200 We the more than min. duration 10 immediately 150 after by the a which applica- irrigation with concentrations in noted 10-100 min ƒÊ after before min recovery of of Within inhibited But range recovery. rate velocity. after concentration About completely again, in concentration did Effect the cells of inhibited considerable by time depended on lag the con- drug. the of 1ƒÊg/ml halt streaming was even in and 50 % normal observed not at the rotational to been their drug inhibition within had complete was did CB CB not of Streaming that the of decreased regained streaming by courses inhibited cells 50 ƒÊg/ml centration time completely solution, 100 ƒÊg/ml reversibly the streaming was before of inhibited 1 shows streaming, was on the streaming with of 1 % DMSO the cell mostly normalized vertical with in against bar is the min (-•œ-), control of this value. of the the no cells treated detectable irregularity or two after the start solution. mean. or The 100 pattern. where rate of CB at min The track a stacked streaming temperature, the (arrow (-•£-). (arrow mean 200 streaming. ƒÊg/ml. measurements µg/ml 60 represents on cell, for at room point its the of velocity treatment 10-20µm/sec Each of with effect in areas (-•¢-) After range S.E. of 50 (-•›-). the the one 30 10 % caused in rate. concentrations was but disordered 40 produced For 2), the the drug immediately was value regarded for five 16 M. Ishigami and R. Nagai mass of cytoplasm, like a helmet, containing chloroplasts and other particles was often formed. This helmet-like mass did not shift position and continued to rotate there at least for few hours (Fig. 2). 2) On the streaming direction: Bundles of microfilaments in Characean cells are not affected structurally by cytochalasin B (1, 14). To obtain information of the structural Fig. 2. nucleus with a) (N), 5 ƒÊg/ml plasts. moved 50ƒÊm, •~ The Normal mesophyll chloroplasts CB. nucleus anticlockwise, 650 The (C) arrow is far but cells and points from the this of other to mass. helmet-like Vallisneria. particles The shows a helmet-like c) The mass thin mass same has layer rotational of cell not (as moved of cytoplasm streaming. cytoplasm in containing b) containing 2b) after from its 10 sec. original The cell several The nucleus position. the treated chlorohas Scale, Motile Apparatus stability of the streaming the on the concentration average plateau reversal (Fig. fact that suggests every cell, dependent the the and that the With Vallisneria drug on of inhibition investigation; The cells 70-80 was were % hibited. of toxic. removal In of Fig. 3. 50 (-•›-). Each Percentage cells more the Effect (-•œ-), of point cells examined than drug (-•¢-) of at and CB or on clear cells. 30 % represents (Ntotai). the the of streaming drug, mean their Abscissa: rate: Lead Duration 100, In the and 100 ƒÊ depending of 50 (Y. 100 ƒÊg/ml, the observed in are the control, the 50 completely of the by CB regenerated in- regenerated acetate micro- 100 five of the or 500 the mM streaming was mM was were samples the for for 1 h. was this not When observed completely (i.e., 200 and was not resumed applied treated of after drug though is useful in- 500 mM) after long. drug direction a reversible examined. streaming cells has cells, action streaming before The also its 200 of cells, died for first 50, than Control value h. of of was cells, direction. streaming 24 internodal cessation treated cells (-•£-). the reversed probability 10, other the and bundles the higher the the reversibility of In of most drug inhibition of 50 was of 50 a plateau of disorganized Characean The a concentration 100 ƒÊg/ml which in (5). of 10, 50 %. streaming 10 mM the the streaming concentrations treated acetate within are of is the mode at with the On reaching direction because polarity the finally 10 ƒÊg/ml removal reversal of concentrations streaming polarity, is not applied at the of 1 % DMSO. increased gradually treatment, at checked concentrations microfilaments cytoplasmic treated Lead the the 1) therefor, drug of and of after acetate. effect mechanism of we at in a solution cells examined treatment bundles cells, treatment duration reversed of lead inhibitory total the 3). original having Effects was and reversal that of filaments in by was reached within 3 h, remained a few percent. The the in caused As a control, cells were immersed ratio of reversed cells to the total on in 17 Cells microfilaments direction g/ml. The cells in Vallisneria about the treatment. at with twenty removal concentrations 1 % cells of the DMSO each. drug of 10 solution Ordinate: (Nx)O to the 18 M. Ishigami and R. Nagai Reversible inhibition was observed with treatments at 50 and 100 mM. Fig. 4 shows the time courses of the inhibition of streaming and its recovery. Streaming in every treated cell was inhibited completely within 30 min at 100 mM, and within 45 min at 50 mM. Disturbances in the streaming track and the stack of cytoplasm, observed with CB treatment, did not occur with lead acetate. Cells inhibited completely at 50 mM resumed their normal velocity within 90 min after the drug had been washed out. The recovery in cells that had been treated at 100 mM was slightly delayed. We noted that streaming started without a time lag after removal of the drug as was observed with CB treatment. Fig. 4.4. Effect Fig. Effect of of lead lead acetate acetate on the on the streaming streaming rate. rate. As in As in the the case case of CB, of CB, lead lead acetate acetate was applied was applied at the at the concentrations concentrations of 50 of 50 (-0—) (-•œ-) or 100 or 100 mM mM (-•¢-). (—A—). Tap Tap water water was was used used as the as the control control medium medium (-0—). Each (-•›-). Each point point represents represents the mean the mean value value for five for five cells. cells. The The length length of the of the vertical vertical bar bar is the is the S.E. S.E. of the of the mean. mean. TABLE 1. EFFECT OF LEAD ACETATE ON THE STREAMING *1 Number of cells which reversed *2 Total number of cells examined streaming . in one sample . DIRECTION Motile Apparatus in Vallisneria 19 Cells 2) On the streaming direction: Effects of the drug at 50 and 100 mM were examined as described above. The treatment periods were set at 1 and 3 h because treating cells for a longer period (such as 1 day) killed even those immersed in rather dilute solutions (i.e., 50 mM). Results are shown in Table 1. Lead acetate produced no reversal of the streaming direction in almost all the cells. This differs sharply from the results obtained for CB treatment, and suggests that the microfilament bundles must be structurally stable during the cessation of cytoplasmic streaming caused by lead acetate. DISCUSSION The present g/ml, but ing locally ceased due of treated the quickly the presence cessation of that peripheral its and served when integrity the by Lead the the of Vallisneria the Based not must integrity does on the to is not that particles directed had cells 3) the caused CB occurred Lead in acetate in of the at 10-100 about at same integrity CB reversal of check the cytoplasmic further of the data extent 50 % 50-100 mM direction the acetate was is strong Characean are cells. in seems microfilament bundles 14). However, not to be be the Our next bundles in is prestructural maintained these the to bundles Characean which This localized there direction. to for as investigated. in CB rigidly labile kept their during step will are broken be streaming the the conclusions microfilaments the simple along those 1). in cessation were the of are no reversion of obtained simple this of cessation the inhibition important is evidence of streaming in Vallisneria the bundles Characean of experiment when darkness are we microfilament as preliminary disorganized stability with mechanism However, bundles; constructed in Vallisneria The for disorganization. treatment, that of our become in is removed. to be closely related partly or completely governs tentative (1, streaming streaming drug microfilament result be so the be not rotational as the is assumed around were with along finally the the The microfilament motile lead to their disinte- the microfilaments in darkness. When light, newly united of concerned, walls that kept cells However, would to regeneration of disordered in darkness. the anticline cells. streaming suggested cells are irradiated in their pericline walls usually moved cytoplasmic tracks. These streamlets factor of of treatment the lead in However, stability Vallisneria and that results). structural during CB microfilaments (unpublished structural to by to bundles the in of been processes seem which become far many The after gration. One result have a tendency to Our direction is homologous between inhibits seem integrity as its drug. by of rotational streamƒÊ 2) When streaming had drug. and hours what inhibited track streaming Vallisneria that ceases direction clear fact microscope acetate about by because streaming in microfilaments electron down steady the removal, mechanism streaming of use streaming Vallisneria. treatment cells of of its completely 5 ƒÊg/ml broke the of the cytoplasm. reversion removal After difference Characeae do CB, the motile region a considerable few was CB at movement of after streaming. is supported not Streaming restored. The to : 1) reversibility. irregular cells halted evidence CB showed good induced to gradually also study with and cell the cytoplasmic formed, and (cf. 12, microfilament We must now diversely established 13). These bundles determine bundles. mechanism is homologous of to secondary primary streaming, streaming once a 20 M. Ishigami and R. Nagai rotational streaming is established. 2). The motile system of secondary streaming differs in the structural stability of the microfilament bundles from that of primary streaming. Cytoplasmic organelles equipped with myosin-like protein need to be investigated further. Myosin-like protein already has been extracted from Elodea and partially purified (11). REFERENCES 1. BRADLEY,M.O. Microfilaments and cytoplasmic streaming: inhibition of streaming with cytochalasin. J. Cell Sci. 12, 327-343, 1973 2. FORDE, J. and M.W. STEER.Cytoplasmic streaming in Elodea. Can. J. Bot. 54, 2688-2694, 1976 3. HAUPT, W. and E. SCHORBOHN.Light-oriented chloroplast movements, in Photobiology of Microorganisms, ed. Halldal, P., Wiley-Interscience, London, pp. 283-307, 1970 4. HAUPTFLEISCH, P. Untersuchungen fiber die StrOmung des Protoplasm in behauteten Zellen. Jb. wiss. Bot. 24, 173-234, 1892 5. KAMITSUBO, E. Physiological studies on the mechanism of cytoplasmic streaming in characean cells. IV. (in Japanese) Proc. 41st Annual Meeting, Bot. Soc. Japan, 71, 1976 6. KAMIYA,N. Protoplasmic streaming. Protoplasmatologia 8, 3a, 1-199, 1959 7. KERSEY,Y.M., P.K. HEPLER,B.A. PALEVITZand N.K. WESSELLS. Polarity of actin filaments in Characean algae. Proc. Natl. Acad. Sci. U.S.A. 73, 165-167, 1976 8. NAGAI, R. Cytoplasmic streaming in plant cells. What's new in Plant Physiology, 9, in press 9. NAGAI,R. and T. HAYAMA.Ultrastructure of the endoplasmic factor responsible for cytoplasmic streaming in Chara internodal cells. J. Cell Sci. 36, 121-136, 1979 10. NAGAI, R. and L.I. REBHUN.Cytoplasmic microfilaments in streaming Nitella cells. J. Ultrastruct. Res. 14, 571-589, 1966 11. OHSUKA,K. and A. INouE. Identification of myosin in a flowering plant, Egeria densa. J. Biochem. 85, 375-378, 1979 12. SEITZ, K. Primary processes cotrolling the light induced movement of chloroplasts. Acta Protozool. 11, 225-235, 1972 13. SEITZ,K. Orientation in space: Plants. in Marine Ecology, Vol. 2, ed. Kinne, 0., Wiley-Interscience, London, pp. 451-497, 1974 14. WILLIAMSON, R.E. A light microscope study of the action of cytochalasin B on the cells and isolated cytoplasm of the Characeae. J. Cell Sci. 10, 811-819, 1972 (Received for publication, November 6, 1979)