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) 8ll Printed in Great Britain

A LIGHT-MICROSCOPE STUDY OF THE ACTION OF CYTOCHALASIN B ON THE CELLS AND ISOLATED CYTOPLASM OF THE CHARACEAE

R.E.WILLIAMSON The Botany School, Downing Street, Cambridge, England

SUMMARY The reversible inhibition of cytoplasmic streaming in cells of Nitella translucent by cytochalasin B was found to be accompanied by an increase in the number of mini-vacuoles in the endoplasm. Fibrils thought to drive the streaming were still present in inhibited cells. In droplets of cytoplasm obtained from cut cells of Chara corralina, the final number of fibrils formed was not sensitive to cytochalasin B, although the motility of these fibrils was highly so. The movement of organelles in the absence of visible fibrils and the rotation of chloroplasts were also inhibited. The evidence for the involvement of the microfilament system in cytoplasmic streaming is discussed. The way in which such microfilaments seem to be operating in characean cells differs from the most likely mechanism for their operation in other cytochalasin- sensitive processes.

INTRODUCTION Many investigations of cytoplasmic streaming in plant cells have made use of the large size and regular streaming pattern of characean cells. In such cells the interface between the stationary cortical gel and the flowing cytoplasm has been identified as the site at which the motive force for the streaming is generated (Kamiya & Kuroda, 1956). An electron-microscope study (Nagai & Rebhun, 1966) revealed bundles of microfilaments, each filament being some 5 nm in diameter. The location of these microfilaments at the interface where the motive force is produced, their orientation parallel to the direction of streaming, and their resemblance to filamentsimplicate d in motile processes in other cells, led to their being favoured as the system responsible for generating the motive force. Fibrils with the position and orientation of the microfilament bundles have been observed with the light microscope (Kamitsubo, 19666). No motion of these interfacial fibrils themselves was seen, but organelle movement was especially rapid along the tracks of the fibrils. Light-microscope studies of protoplasmic droplets from cut characean cells (Jarosch, 1956a, b, 1957, 1958; Kamiya, 1962; Kuroda, 1964) and of centrifuged cells (Kamitsubo, 1966a, b) showed the presence of motile fibrils. Electron microscopy of the protoplasmic droplets (Rebhun, 1967) demonstrated the presence of microfilament bundles which, it was thought, represented the motile fibrils. On the basis of its activities in a wide variety of cells, it was proposed that cyto- 52-2 812 R.E. Williamson chalasin B was a specific inhibitor of the functioning of microfilaments (Wessels et al. 1971). Their finding of the sensitivity of cytoplasmic streaming to cytochalasin B greatly strengthens, therefore, the case for the role of microfilaments in generating the motive force. In this paper, further observations on the action of cytochalasin B on characean cells are presented, and the effect of the drug on the behaviour of protoplasmic droplets is described.

MATERIALS AND METHODS Materials Cytochalasin B was obtained from I.C.I. Pharmaceuticals Division, Alderley Park, Cheshire. A stock solution in dimethyl sulphoxide (Carter, 1967) of 5 mg per ml was prepared, and appropriate dilutions made with distilled water. For all control experiments, equally diluted dimethyl sulphoxide (DMSO) was used. Characean cells were obtained from laboratory cultures. For studies with intact cells, small leaf, internodal and rhizoidal cells of Nitella translucens were used most extensively. For obtaining isolated protoplasm, large internodal cells of Cliara corralina were commonly used.

Microscopy A Zeiss Universal Research Microscope with differential interference-contrast optics was used for all observations.

Intact cells The cell was mounted in the control solution of dimethyl sulphoxide, the coverslip being supported at each corner with a small piece of caulking compound. Filter-paper strips were used to draw in cytochalasin B, and later to wash the drug out. The velocity of cytoplasmic streaming was obtained by measuring, with a stop-watch, the time for organelles in the endoplasm to travel distances measured with a micrometer eyepiece. The average of several measurements was taken. The number of mini-Vacuoles (Costerton & MacRobbie, 1970) in the endoplasm passing through the field of view in a measured time was recorded with a hand counter. As the velocity of streaming was known, this figure could be converted into the number of mini-Vacuoles present in a strip of endoplasm 1 mm long and 100/tm wide. The values plotted are, where possible, the mean of two or more determinations.

Isolated cytoplasm The end of a cell was cut off with scissors and the cell contents expelled by squeezing between thumb and finger. The contents of several such cells were used for each experiment. A shallow ring of Vaseline was placed on a microscope slide and 5 [A of cytochalasin B, or of the control DMSO solution, pipetted into the ring; 95 fil of the expressed cell contents were added and mixed by gently blowing through the pipette. The cytochalasin levels mentioned in the results refer to the final concentrations. A coverslip was placed on the ring of Vaseline, and excess Vaseline and cell contents expressed by gentle downward pressure. Timing of the experiment began immediately after the completion of these steps, when the slide was transferred to the microscope. In sampling experiments, periods of 10 or 15 min were used, some 15 of the droplets of protoplasm in the preparation being observed in each period. The presence or absence in each of the droplets of fibrils and of various forms of motility were recorded, and the number of vacuoles in each droplet counted. The forms of motility scored in this way are described in the Results section. Action of cytochalasin B on Characeae

RESULTS Intact cells The inhibition of streaming. The reversible inhibition of cytoplasmic streaming was observed with cytochalasin levels of from 1-50 /ig/ml. The response with the higher levels occurred in a few minutes (Fig. 1 A), while 1 /ig/ml levels gradually produced a similar inhibition over about 6 h. The inhibition was rapidly reversed on washing out the cytochalasin with the control DMSO solution (Fig. 1 A), although the inhibi- tions produced by higher concentrations of the drug were irreversible after about 60 min.

25 ,«g/ml Control solution of cytochalasin B Control solution dimethyl sulphoxlde | I of dimethyl sulphoxide

80 120 160 200 240 Time, min Fig. 1. The effect of a 30-min treatment with cytochalasin B (25 fig/ml) on an internodal cell of Nitella translncens. A, The velocity of streaming; B, The frequency of mini-vacuole8 in the endoplasm.

In several favourable cells interfacial fibrils resembling those described by Kamit- subo (1966&) were observed. Although endoplasmic organelles moved extremely rapidly when close to such fibrils, no oscillatory movement of the fibrils was seen. Such fibrils were still present in cells in which streaming had been halted by treatment with cytochalasin B. Changes in tlie endoplasm. A reversible increase in the frequency of mini-vacuoles 814 R. E. Williamson was observed in a majority of the cytochalasin-treated cells (Fig. i B). Their rate of appearance and reversibility was similar to the behaviour of the rate of streaming at the various cytochalasin levels used. The presence of certain areas with large numbers of mini-vacuoles and their virtual absence from other areas accounts for the high scatter of the points during the cytochalasin treatment. A similar tendency to appear in groups was noted for the mini-vacuoles of untreated cells. In addition to an increased number of mini-vacuoles caused by cytochalasin B, the upper size limit of the individual mini-vacuoles was often noticeably increased. During rapid swelling of vacuoles, fusion between adjacent ones was observed in some cases.

Exuded cytoplasm General appearance. Approximately spherical droplets containing numerous organelles collected at the bottom of the shallow chamber, whose walls were formed by the Vaseline ring. The diameters of the droplets ranged from about 10 to 80 fim. They contained all the organelles identifiable in the intact cell. Outside the droplets, the much more dispersed organelles did not usually show movement other than what was assumed to be Brownian motion. However, in freshly exuded material, long linear fibrils resembling the interfacial fibrils of the intact cell were occasionally observed. Without any visible motion of the fibril, several spherosomes were seen to move in one direction along its surface. Observations were carried out on the droplets over a 2-h period, since by the end of that time motility was more or less absent (Table I,A), and signs of degenera- tion such as the appearance of myelin-type figures and the coagulation of organelles were apparent. Motility in the droplets. Various forms of motility were recognized in the droplets: (a) Some regions of cytoplasm showed discontinuous movements of a few /tm, in which all the organelles in the area took part. The same region might shortly after- wards show a similar movement in a different direction. In addition, individual organelles in such areas sometimes showed saltatory movements independently of neighbouring organelles, some of which might be moving simultaneously in other directions. Certain regions of a droplet might be persistently more active than neighbouring areas, but no fibrils were visible to distinguish such active areas from neighbouring, less active areas. (b) A variety of fibrillar structures, many of which showed motility, were observed in the droplets. The various forms of movement which such fibrils can undergo have been described in other studies (Jarosch, 19560,6, 1957, 1958; Kamiya, 1962; Kuroda, 1964). They were observed with free ends and as closed loops, some of the latter assuming the shapes of regular polygons and rotating. Some fibrils showed flexuous movements suggestive of wave propagation, but the movement of others around their droplet occurred in the absence of visible deformation of the fibrils. A single droplet might possess several fibrils. The affinity of small spherosomes for the fibrils has been described (Kamiya, 1962). Such organelles appeared to be fixed to the fibrils and served as markers to pick out very fine, moving fibrils. Other organelles near to moving fibrils were often seen to Action of cytochalasin B on Characeae 815 move in the opposite direction to that in which the fibril was travelling (Jarosch, 1956a). (c) Many of the chloroplasts within the droplets showed rotation, and a counter- stream of organelles moving in the opposite direction to that of the chloroplast was frequently seen. No rotation of nuclei or other organelles in the droplets was seen, and in the intact cell only the few chloroplasts travelling in the endoplasm were observed to rotate. Previous studies of both intact cells and cytoplasmic droplets have shown that both chloroplasts and nuclei may rotate (Kamiya, 1962, and references therein; Kuroda, 1964). No rotation of any chloroplasts outside the droplets was observed in these experiments. Changes occurring in the droplets with time, and the effects of cytochalasin B. The

Table 1. The percentages of droplets {with standard errors of the mean) in control and cytochalasin-treated preparations showing various forms of motility and fibrils

Time, min A 0-15 3O-45 65-80 105-120 A. Droplets showing any form of motility Control 79 ±10 69 ±3 45 ±6 4±2 0-2 /ig/ml I9±4 i6±9 n±3 4±2 cytochalasin B B. Droplets with moving organelles but no visible fibrils Control 45 ±9 i7±5 8±4 3±2 02 /fg/ml 8±2 n±5 4±3 I ±1 cytochalasin B Time, min

0^10 20—30 50-60 IIO-I2O C. Droplets showing rotating chloroplasts Control i5±7 n±5 io±5 O 1 /tg/ml 3±2 0 I±I I±I cytochalasin B Time, min

0-15 15-30 45-60 IO5-I2O D. Droplets showing motile fibrils Control 43 ±10 49 ±6 57±7 3 + 2 02 /ig/ml 5±4 I2±7 I2±9 3±2 cytochalasin B E. Droplets showing stationary fibrils Control 0 0 i7±5 38 ±11 0-2 /^g/rnl 4±3 i6± 10 40 ±6 4O±7 cytochalasin B F. Droplets showing any fibril Control 43 ±10 5i±7 57 ±7 41 ± 12 0-2 /tg/ml 9±6 2I±7 44±9 43 ±7 cytochalasin B 816 R. E. Williamson sampling procedure (see Materials and Methods) was used to measure the frequency of the 3 forms of motility previously discussed. A continuous decline in the percentage of the droplets showing any form of motility was observed (Table I,A; control figures). A similar pattern was found for the frequency with which motility in the absence of visible fibrils (Table I,B) and rotating chloroplasts (Table i,c) were observed. In contrast, the percentage of droplets in which motile fibrils could be seen initially rose before falling to a very low level (Table I,D). Stationary fibrils were absent initially, but rose over the period in which the frequency of motile fibrils was declining (Table I,E). The total number of visible fibrils, motile and stationary, increased (Table I,F). Table 2. The number of vacuoles per droplet

Time, min A

O-IO 30-40 70-80 110-120 Control o-8±o-2 i-6±o-7 2-3 ±o-8 o-9±o-3 1 /ig/ml 1-910-9 2-6±o-9 5-1 ± i-o 4-8 ±0-9 cytochalasin B

These features were further clarified by studying continuously individual droplets which did not initially contain any visible fibrils, nor any groups of spherosomes whose movement suggested the presence of very fine fibrils. In many of these droplets, motile fibrils were subsequently found, usually after about 30 min, and became increasingly conspicuous before finally becoming non-motile. In such droplets, the movement of whole areas of cytoplasm, with saltatory movements of individual organelles, was most marked in the early stages of observation. Most droplets which subsequently developed fibrils then showed only the forms of motility normally associated with such fibrils. However, a minority continued in addition to show the initial pattern of motility in areas from which the fibrils were absent. The sampling technique showed that both of the levels of cytochalasin B used (0-2 and i-o/ig/ml) rapidly produced a strong inhibition of motility in the treated droplets (Table I,A). The inhibition was slightly greater with the higher concentration. It was found that all three of the categories of motility recognized were susceptible to the inhibitor (Table I,B-D). Treatment with cytochalasin led to non-motile fibrils appear- ing in some of the droplets in the first hour, when such fibrils were absent from the control (Table I,E). The percentage of the treated droplets showing any fibrils, motile or non-motile, was initially lower than for the control droplets, but the final percentage was the same in both cases (Table I,F). The number of vacuoles per droplet in the control preparations showed a gradual rise over the first hour, subsequently declining somewhat (Table 2). Although the figures for the number of vacuoles in all of the sampling periods using 0-2 /tg/ml cytochalasin B exceeded the corresponding figures for the control experiments, it was only with the higher level of the inhibitor that a reliable separation between the 2 sets of figures was obtained (Table 2). It was noticeable that the increased value for the Action of cytochalasin B on Characeae 817 number of vacuoles per droplet was largely the result of the presence of some droplets with very large numbers.

DISCUSSION The observations, both with intact cells and isolated cytoplasm, have shown that the formation of additional mini-vacuoles could be induced by cytochalasin B. At all the concentrations used with intact cells, the appearance of the mini-vacuoles was as rapid as the decline in the rate of streaming. In the case of the protoplasmic droplets, the large increase in the number of vacuoles with a cytochalasin level of 1 /

1 am grateful to Dr E. A. C. MacRobbie for much advice and assistance during the course of this work, and also to the Science Research Council for financial support.

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