Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of rrprodwtion in any form resmord
Experimental
INDUCTION
Cell Research 90 (1975) 119-126
OF DIVISION
TETRAHYMENA
SYNCHRONY
IN
PYRIFORMIS
A Pressure Study A. M. ZIMMERMAN Department
of Zoology,
University
and HELEN of Toronto,
L. LAURENCE
Toronto,
Ontario,
Canada
MSS
IA1
SUMMARY Division synchrony was induced in Tetrahymena pyriformis by subjecting log growth cells (at 28°C) to an alternating series of hydrostatic pressure pulses separated by 30 min recovery periods at atmospheric pressure. The division maximum, approx. 50 % in growth medium (PPL), occurred at 80 min after the last pressure pulse (EP), the second synchronous division 30 % occurred at 210 min after the last pressure pulse (EP). RNA, DNA and protein synthesis occurred throughout the period from the end of the last pressure pulse to the first synchronous division. At the time of first division there is a reduction in the net increase of W-thymidine incorporation; after a short interval of time there is a resumption of thymidine incorporation. The G 1 interval of the second division cycle accounted for approx. 29 % of the cell cycle. The mechanism for pressure induced division synchrony are discussed in the terms of the known action of pressure on Tetrahymena.
Populations of Tetrahymena pyriformis can be induced to divide synchronously following treatment by a variety of physical and chemical agents. The best known and most widely used technique is that of Scherbaum & Zeuthen [18] using cyclic heat shocks. Since this method was first reported it has been extensively used to synchronize cells which were employed as models for studies on cytokinesis. Most recently this technique has been modified so that DNA synthetic events as well as furrowing are synchronous [28]. Other methods successfully employed for synchronizing Tetrahymena are the cold thermal treatment [16], starvation refeeding method [l], thymidine deprivation [22], vinblastine [19] and the use of microtubule disrupting alkaloids, colchicine and Colcemid [24]. Most recently Wolfe [23] has reported a new procedure for selection of synchrony using
gradient centrifugation. Procedures for synchronizing Tetrahymena have been extensively reviewed [2, 17, 26, 291. Hydrostatic pressure is known to alter the cyclic activity of dividing cells. Short pulses of pressure differentially extend the division schedule in marine eggs [32], and Tetrahymena [9, 201. Hydrostatic pressure can disrupt the integrity of the mitotic apparatus as well as specifically inhibit cytokinesis. The conformational changes in structural proteins which result from the pressure treatment may be responsible for these disruptive effects [5, 6, 14, 211. Synthesis of ‘division-essential’ material can also be reduced by pressure treatment (cf reviews [7, 30, 31, 331). Since it has been proposed that thermal and chemical treatment induce synchrony through modification of synthetic events within the cell cycle, it is conceivable that high pressure Exptl
Cell Res 90 (1975)
120
Zimmerman
and Laurerzce
could also be employed to induce division synchrony. The following studies, concerning the influence of pressure on log growth cultures of Tetrahymena, centre on the following questions: whether hydrostatic pressure can be employed to synchronize Tetrahymena, and whether macromolecular synthesis in these pressure-treated cells is similar to heat synchronized cultures.
MATERIALS
AND
METHODS
Organism Tetrahymena pyriformis CL stock cultures, originally a gift from the late Dr 0. Scherbaum, were maintained axenically at 28°C in a PPL nutrient medium (2 “0 proteose peptone supplemented with a 0.1 % liver fraction “L”).
Pressuresynchronization technique Flasks containing 100 ml of PPL were inoculated with l-3 ml of 2-3 day old culture. These cells were grown logarithmically (at 28°C) for 15 h and then subjected to pressure pulses Cells were placed in a lucite chamber (72 ml); covered with parafilm and placed into a stainless steel pressute chamber (at 28°C). The cell density (30 00&50 000 cells/ml) was measured with a Model Z Couher Counter fitted with a 200 hum aperture tube. The temperature-pressure apparatus was patterned after one originally designed by Marsland [12] (cf [31]). Pressure up to 14 000 psi was attained at the rate of 5 000 usiistrokelsec. Various magnitudes of pressure, 5 OO&ldOOO psi (equivalent t;o 344.5 ): lo5 - 689 x IO6 N/m2 or Pascals) were aaolied for durations of l-4 min; the pressure was released virtually instantaneously by means of a needle value. The cells were transferred to flasks and placed into a shaker bath at 28°C. It required 2 min to disassemble the pressure apparatus. The pressure pulse treatment was repeated 5-7 times. A standardized recovery period of 30 min was maintained between pressure pulses. The end of the last pressure treatment was designated EP. Ceil samples (at 28°C) were removed from the flasks at various times after EP and the ‘division index’ (i.e. the % cells showing cytoplasmic furrows at any given time) and cell density were determined. The division maximum, occurring at a time when the majority of cells displayed cytoplasmic furrowing, was determined by plotting the changes in the division index as a function of time.
Biochemical procedures In the isotope centrifuged and prior to the last were added to Exptl
incorporation studies the cells were resuspended in inorganic medium [3] pressure pulse. At 2 min EP, the cells a flask containing one of the radio-
Cell Res 90 (1975)
active precursors (final concentrations: 0.3 pCi/ml W-thymidine, 2.5 &i/ml 3H-uridine, or 0.05 &i/ml “C-phenylalanine). At appropriate times, duplicate 0.5 ml aliquots were removed and immediately placed in an ice bath. An equal volume (0.5 ml) of cold trichloroacetic acid (TCA) was added to each aliquot (10 % TCA for DNA and protein determinations and 20 qb TCA for RNA determination). The aliquots were then allowed to remain at 0°C for 12 h. For phenylalanine incorporation studies, the samples were subjected to an additional procedure; the samples were placed in a bath of 90°C for 15 min. The radioactive material was collected on glass fiber filter pads (Reeve Angel, Clifton, N.J.) and washed with TCA, 95 and 100 % ethanol. The dried filter pads were placed in scintillation vials containing 15 ml of scintillation fluid (5.5 g of Permablend I (Packard Instrument Co. Inc., Downers Grove, Ill.) and toluene to make 1 liter). The radioactivity was measured with a Packard Tri-Carb liquid scintillation spectrometer. The counting efficiency was 33 “b for 3H and 85 :,, for ldC.
Both 5-3H-uridine (spec. act. 20.0 Ci/mM) and 2-Wthymidine (spec. act. 51.8 mCi/mM) were obtained from Schwarz BioResearch, Orangeburg, N.Y. L-3l*C-phenylalanine (spec. act. 15.3 mCi/mM) was purchased from Amersham/Searle Corporation, Arlington Heights, Ill., USA.
RESULTS Preliminary observations It has been previously reported that high pressureblocks cytokinesis and that moderate pressuresdelay division in heat synchronized Tetrahymena [9]. Log growth phase Tetrahymena are similarly affected by high hydrostatic pressure [ 111. The effects of high pressure on log cells vary in severity depending upon the magnitude of the pressure and the duration of the pressure treatment. High pressures (10 000 psi) for a long duration (10 min) drastically affect logarithmic growing cells causing them to become round-this is accompanied by a cessation of forward movement, although uncoordinated ciliary activity continues. A short time (5-10 min) after decompression most of the cells show some slow movement; they retain their roundish appearance and display prominent cytoplasmic vacuoles.
Pressure-induced
division synchron)!
121
Fig. I. Abscissa: duration of pressure (min); ordinate:max. % dividing. 0, 5 pressure pulses; m, 7 pressure pulses. Induction of division synchrony as influenced by duration of pressure, number of pressure pulses and magnitude of pressure. In all cases the cells were subjected to compression in PPL growth medium for a specified duration (l-4 min) and permitted 30 min for recovery between pulses in a shaker bath at 28°C. Under certain specified pressure-duration studies multiple division maxima were recorded, these are shown as irregular profiles at 7 500 psi for 3 and 4 min and at 10 000 psi for 1 min. Optimum synchrony was attained following 7 pulses at 7 500 psi for 2 min duration, and following 5 pulses at 8 500 psi for 2 min duration.
Lower magnitudes of pressure (7 500 and 8 500 psi) for short durations (l-4 min) did not induce major changes in the shape or activity of the cells. The posterior portion of some cells began to round 2 min after compression and this shapechange was more pronounced 4 min after compression. Ciliary velocity was also slightly reduced. Upon decompression the cells exhibited a rapid increase in movement; within 2 min the compressedcells displayed movement which was comparable to non-pressurized control cells. Previous studies [ 151 demonstrated that following pressure treatment abnormal patterns of nuclear morphology were observed when cells were allowed to remain in their culture flasks without agitation. The aberrations were attributed to 0, deprivation following the pressure treatment; nuclear abnormalities were not evident in pressurized cells that were subjected to gentle agitation following decompression.Thus the subsequent experiments were designedto employ moderate pressuresfor short durations and to in-
elude gentle agitation during the periods between pressuretreatments. Induction of synchrony From the foregoing observations it was evident that in order to induce synchrony it would be necessaryto usea pressure-duration combination which would produce a significant division delay but would not markedly affect cellular shape and activity. Since Scherbaum & Zeuthen [18] have established that a high degree of synchrony is induced following a series of cyclic temperature shocks at 34°C for a duration of 30 min followed by a recovery period of 30 min at 28°C a series of experiments were designed in which a duration of pressure treatment would be kept short and the recovery time would be kept constant (30 min). Various levels of hydrostatic pressure (5 000, 7 500, 8 500 and 10 000 psi) for different durations (l-4 min) were applied systematically 5 to 7 times. At both the low (5 000 psi) and the high (10 000 psi) pressuresit was not Exptl
Cell
Rrs 90 (1975)
122 Zimmerman
and Laurence
Fig. 2. A series of photomicrographs of Tefrahymena pyriformis prior to and after pressure induced synchrony. Division synchrony was induced by subjecting log growth cells (at 28°C) to a series of 5 pressure pulses, (8 500 psi for 2 min duration); each pressure pulse was separated by 30 min at atmospheric pressure (at 28°C). (a) Log growth cells; (b) cells 11 min after the last pressure pulse; (c) Tetrahymena cells 80 min after the last pressure pulse; (d) most of the cells in the field at 85 min after the last pressure pulse have undergone division.
possible to induce synchronous division (fig. 1). At the 5 000 psi level, the cells continued to divide and the division indiceswere comparable to control cells at atmospheric pressure. Whereas, at the 10 000 psi level, the cells developed abnormal shapes and displayed irregular swimming activity. A treatment of 10 000 psi for 1 min applied 5 times resulted in small multiple bursts of synchronous division (18 y0 divided at 80 min after the last pressure pulse (EP), 14 % divided at 100 min EP, 10 % divided at 110 EP and 8 % divided at 120 min EP); in these experiments division Exptl
Cell
Res 90 (1975)
index was employed as a criteria for synchrony. A relatively high degree of synchrony (approx. 50 %) was routinely attained at pressuresof 8 500 psi for 2 min (cf fig. 2) and at 7 500 psi for 2 min. The optimum synchrony was attained at 7 500 psi after a series of 7 pressure pulses, whereas a comparable synchrony was attained at 8 500 psi after 5 pressure pulses. Increasing the number of pressure pulses at 8 500 psi to 7 did not increase the division synchrony. The first division peak occurred at 65-70 min and
Pressure-induceddivision synchrony
123
Cell counts taken during the experiments showed a doubling of cell population after the first synchronous division and again a further increase after the second division. Log growth-phase cells at atmospheric pressure were monitored as controls. The size and shape of the pressure-synchronized cells were two to three times as large as log growth phase Tetrahymena and appeared similar to heat synchronized cells.
5oi 401
301 J70 20 50
.Macromolecular synthesis
101
430
I40
80
120
160
200
240
280
3. Abscissa: time after EP (min); ordinate: (left) ‘% dividing (O-O); (right) cells/ml x 1OW. The development of division maximum in pressure synchronized Tetruhymena. Cells in PPL growth medium were pulsed 5 times at 8 500 psi for 2 min duration; each pressure pulse was separated by 30 min at atmospheric pressure. First and 2nd division peaks occurred at 80 and 220 min, respectively, after the last pressure pulse. The cell densities of pressuretreated cells ( x . . . x ) and log growth cells (A--A) are shown. Fig.
75-80 min EP at 7 500 and 8 500 psi, respectively. The second division peak (approx. 30 %) occurred approx. 210-230 min after the last pressuretreatment at either pressurelevel (fig. 3).
{E-40
80
The incorporation of 14C-thymidine, 3Huridine and r4C-phenylalanine into the TCAprecipitable material was employed as an index of DNA, RNA and protein synthesis. Cells were transferred to inorganic medium prior to the last pressure pulse and treated with one of the radioactive precursors immediately after the last pressure pulse (2 min EP). As early as 10 min after the last pulse of pressure incorporation of specific radioactive precursors was determined in the acid-insoluble fractions. Between 20 and 40 min EP there was approximately a 2-fold increase in accumulative incorporation of 14Cthymidine and 14C-phenylalanine; and approximately a 50% increase in 3H-uridine incorporation (fig. 4). It should be noted that
i[l(K 120
I-I/
160
200
240
280
40
80
120
40
80
120
time after EP (min); ordinate: cpm x lo+. The incorporation of W-thymidine, SH-uridine (cpm [ x lO-2] per 8 000 cells) and %-phenylalanine (cpm [ x lo-*] per 8 000 cells) into DNA, RNA and protein respectively. Division synchrony was induced by a series of 5 pressure pulses of 8 500 psi for 2 min duration. Tetruhymenu were continuously exposed to the appropriate radioisotope commencing immediately after the last pressure pulse and the incorporation into the TCA-insoluble material was determined at various intervals thereafter. The incorporation of W-thymidine into pressure treated (O-O) and heat-treated ( x --- x ) cells are compared. The cell density of pressure treated and heat treated cells following treatment was 10 000 cells/ml and 12 000 cells/ml respectively; duplicate samples of 0.5 ml aliquots were removed for radioisotope determination (see text for details). Fig. 4. Abscissa:
Exptl
Cell Res 90 (1975)
124 Zimmerman
and Laurence
although the time for maximum division in inorganic medium was consistently delayed by 25 min, the division maximum was not decreased. DISCUSSION The present study confirms the prediction originally proposed by Dr Erik Zeuthen in 1957, namely, hydrostatic pressure treatments could be substituted for temperature treatments as a method for inducing synchronous division in Tetrahymena. Short pulses of hydrostatic pressure systematically applied to cells induce log growth phase cultures to divide synchronously. The division schedule and macromolecular synthesis in pressure synchronized Tetrahymena are similar to those found in heat synchronized cells. The increase in cell density of pressure synchronized cells compares favourably with the cell density increase of heat synchronized cells. The mechanism by which high pressure induces division synchrony in Tetrahymena pyriformis is subject to speculation, however, the hypothesis proposed by Zeuthen (cf [26]) concerning the synthesis of ‘division-essential proteins’ remains most attractive and the present studies support the hypothesis that the synthesis of essential material is altered by the physical effects of high pressure. The division maximum of pressure synchronized cells was approx. 50 % compared to a division maximum of 80-85 % for heat synchronized cells. The schedule of the first division (75 to 80 min E.P.) which resulted from the repetitive pulses of 8 500 psi, was comparable to that found with heat synchronized cultures. However, at lower magnitude of pressure (7 500 psi) the first division peak occurred approx. 10 min earlier. In general the duration of the second division cycle (145 min) of pressure-induced cells was greater than that found in heat-synchronized cultures (100-110 min). It is of interest to Exptl
Cell Res 90 (1975)
speculate that the small peak frequently observed in pressure synchronized cells appearing approx. 30 min after the major synchronous division index peak is similar to that reported by Zeuthen [27] in heat synchronized cells and may be comparable to the early and late participants in synchronous division. Zeuthen [27] has proposed that heat induces synchronous division in Tetrahymena by a differential extension of the G2 phase. Although no direct evidence is available, the similarity of the pressure-synchronized cells to heat-synchronized cells suggests a similar mechanism may be involved. Recently Zeuthen [28] has reported repetitive synchrony in Tetrahymena following heat shocks spaced a normal cell generation apart (160 min). In this technique for synchrony, each heat shock is initiated at the time the population has almost passed through the S phase, thus the heat shocks affect early G2. The display of free running synchronous divisions yield favourable division synchrony and replication. Systematically applied pressure pulses induce a high degree of division synchrony, however, DNA synthesis during the period from the last pressure pulse to the first synchronous division is not synchronized. RNA, DNA and protein synthesis continues throughout this same period, a pattern also reported for heat synchronized Tetrahymena. Although autoradiographic studies are essential to establish precisely the cycle stages in pressure synchronized cells, some preliminary information can be obtained from analysis of total incorporation profiles. The duration of G 1 in the second division cycle of pressure synchronized cells (in inorganic media) is comparable to the Cl of heat synchronized cells (in inorganic media). The Gl was estimated by measuring the length of time during which there was no net increase in the incorporation of thymidine. The Gl interval
Pressure-induced accounts for approx. 29 “/b of the second division cycle. The mechanism by which pressure induces division synchrony is subject to speculation. Although it is not possible to ascribe division synchrony to any single event, several morphological and biochemical events are implicated in the induction of division synchrony in Tetrahymena. Hydrostatic pressure induces morphological alterations, affects nuclear activity, and differentiation of the oral structure [13, 15, 201. Analysis of fine structure indicates that pressure reversibly disrupts microtubular elements in TetrahJjmena [6] as well as labile structures in other cell systems [31, 331. Biochemical studies have shown that transcription and translation in Tetrahymena are sensitive to hydrostatic pressure. Although pressure reduces the protein synthesizing capacity of cells by disrupting the polysomes [4], ribosomes isolated from pressure treated cells are able to synthesize polyphenylalanine as effectively as ribosomes prepared from non-pressure treated cells [8]. DNA synthesis [15], RNA synthesis and protein synthesis [lo] are reduced in cells following compression. Reduction in the synthesis of ribosomal precursor RNA and heterogeneous high molecular weight RNA are also found following compression [25]. In general, the present study supports the hypothesis that the prerequisites for cell division consist of a definitive sequence of biochemical events coupled with the formation of structural elements. Moreover the differential sensitivity of ‘division-essential’ materials to hydrostatic pressure results in the induction of synchronous division.
This paper is dedicated to Dr Erik Zeuthen, Carlsberg Foundation Biological Institute, who predicted in 1957 the feasibility of employing hydrostatic pressure for induction of division synchrony. This investigation was supported by the National Research Council of Canada.
division synchrony
125
REFERENCES 1. Cameron, I L & Jeter, J R, J protozool 17 (1970) 429. 2. Cameron, I L & Padilla, G M, Cell synchrony. Studies in biosynthetic regulation (ed I L Cameron & G M Padilla). Academic Press, New York (1966). 3. Hamburger, K & Zeuthen, E, Exptl cell res 13 (1957) 443. 4. Hermolin, J & Zimmerman, A M, Cytobios 3 (1969) 247. 5. Johnson, F H & Eyring, H, High pressure effects on cellular processes (ed A M Zimmerman) p. I. Academic Press, New York (1970). 6. Kennedy, J R & Zimmerman, A M, J cell biol 47 (1970) 568. 7. Landau, J V, High pressure effects on cellular processes (ed A M Zimmerman) p. 45. Academic Press, New York (1970). 8. Letts, P J & Zimmerman, A M, J protozool 17 (1970) 593. 9. Lowe-Jinde, L & Zimmerman, A M, J protozool 16 (1969) 226. IO. -Ibid 18 (1971) 20. 11. Macdonald, A G, Exptl cell res 47 (1967) 569. 12. Marsland, D, J cellular camp physiol 36 (1950) 205. 13. Moore, K C, J ultrastruct res 41 (1972) 499. 14. Morita, R Y & Becker, R R, High pressure effects on cellular processes (ed A M Zimmerman) p. 71. Academic Press, New York (1970). 15. Murakami, T H & Zimmerman, A M, Cytobios 7 (1973) 171. 16. Padilla, G M & Cameron, 1 L, J cell camp physiol 64 (1964) 303. 17. Padilla, G M, Whitson, G L & Cameron, I L, The cell cycle. Gene-enzyme interactions (ed G M Padilla, Cl L Whitson & 1 L Cameron). Academic Press, New York (1969). 18. Scherbaum, 0 H & Zeuthen, E, Exptl cell res 6 (1954) 221. 19. Sedgley, N N & Stone, G E, Exptl cell res 56 (1969) 174. 20. Simpson, R E & Williams, N E, J exptl zoo1 174 (1970) 85. 21. Suzuki, K & Taniguchi, Y, Symp sot exptl biol 26 (1972) 103. 22. Villadsen, 1 S & Zeuthen, E, Exptl cell res 61 (1970) 302. 23. Wolfe, J, Exptl cell res 77 (1973) 232. 24. Wunderlich, F & Peyk, D, Exptl cell res 57 (1969) 142. 25. Yuyama, S & Zimmerman, A M, Exptl cell res 71 (1972) 193. 26. Zeuthen, E, Synchrony in cell division and growth (ed E Zeuthen) p. 99. Interscience, New York (1964). 27. - Exptl cell res 61 (1970) 3 11. 28. - Ibid 68 (1971) 49. 29. - Cell cycle controls (ed G M Padilla, I L Cameron & A M Zimmerman) p. 1. Academic Press, New York (1974). 30. Zimmerman, A M, The cell cycle. Gene-enzyme interactions (ed G M Padilla, G H Whitson & I L Exptl
Cell Res 90 (1975)
126 Zimmerman
and Laurence
Cameron) p. 203. (1969). 31. - Int rev cytol 30 32. Zimmerman, A M 38 (1964) 454. 33. Zimmerman, S B
Exptl
Cell
Res 90 (1975)
Academic Press, New York (1971) 1. & Silberman, L, Exptl cell res & Zimmerman,
A M, High
pressure effects on cellular processes (ed A M Zimmerman) p. 179. Academic Press, New York (1970). Received May 20, 1974 Revised version received July 12, 1974
Experimental
INDUCTION
Cell Research 90 (1975) 119-126
OF DIVISION
TETRAHYMENA
SYNCHRONY
IN
PYRIFORMIS
A Pressure Study A. M. ZIMMERMAN Department
of Zoology,
University
and HELEN of Toronto,
L. LAURENCE
Toronto,
Ontario,
Canada
MSS
IA1
SUMMARY Division synchrony was induced in Tetrahymena pyriformis by subjecting log growth cells (at 28°C) to an alternating series of hydrostatic pressure pulses separated by 30 min recovery periods at atmospheric pressure. The division maximum, approx. 50 % in growth medium (PPL), occurred at 80 min after the last pressure pulse (EP), the second synchronous division 30 % occurred at 210 min after the last pressure pulse (EP). RNA, DNA and protein synthesis occurred throughout the period from the end of the last pressure pulse to the first synchronous division. At the time of first division there is a reduction in the net increase of W-thymidine incorporation; after a short interval of time there is a resumption of thymidine incorporation. The G 1 interval of the second division cycle accounted for approx. 29 % of the cell cycle. The mechanism for pressure induced division synchrony are discussed in the terms of the known action of pressure on Tetrahymena.
Populations of Tetrahymena pyriformis can be induced to divide synchronously following treatment by a variety of physical and chemical agents. The best known and most widely used technique is that of Scherbaum & Zeuthen [18] using cyclic heat shocks. Since this method was first reported it has been extensively used to synchronize cells which were employed as models for studies on cytokinesis. Most recently this technique has been modified so that DNA synthetic events as well as furrowing are synchronous [28]. Other methods successfully employed for synchronizing Tetrahymena are the cold thermal treatment [16], starvation refeeding method [l], thymidine deprivation [22], vinblastine [19] and the use of microtubule disrupting alkaloids, colchicine and Colcemid [24]. Most recently Wolfe [23] has reported a new procedure for selection of synchrony using
gradient centrifugation. Procedures for synchronizing Tetrahymena have been extensively reviewed [2, 17, 26, 291. Hydrostatic pressure is known to alter the cyclic activity of dividing cells. Short pulses of pressure differentially extend the division schedule in marine eggs [32], and Tetrahymena [9, 201. Hydrostatic pressure can disrupt the integrity of the mitotic apparatus as well as specifically inhibit cytokinesis. The conformational changes in structural proteins which result from the pressure treatment may be responsible for these disruptive effects [5, 6, 14, 211. Synthesis of ‘division-essential’ material can also be reduced by pressure treatment (cf reviews [7, 30, 31, 331). Since it has been proposed that thermal and chemical treatment induce synchrony through modification of synthetic events within the cell cycle, it is conceivable that high pressure Exptl
Cell Res 90 (1975)
120
Zimmerman
and Laurerzce
could also be employed to induce division synchrony. The following studies, concerning the influence of pressure on log growth cultures of Tetrahymena, centre on the following questions: whether hydrostatic pressure can be employed to synchronize Tetrahymena, and whether macromolecular synthesis in these pressure-treated cells is similar to heat synchronized cultures.
MATERIALS
AND
METHODS
Organism Tetrahymena pyriformis CL stock cultures, originally a gift from the late Dr 0. Scherbaum, were maintained axenically at 28°C in a PPL nutrient medium (2 “0 proteose peptone supplemented with a 0.1 % liver fraction “L”).
Pressuresynchronization technique Flasks containing 100 ml of PPL were inoculated with l-3 ml of 2-3 day old culture. These cells were grown logarithmically (at 28°C) for 15 h and then subjected to pressure pulses Cells were placed in a lucite chamber (72 ml); covered with parafilm and placed into a stainless steel pressute chamber (at 28°C). The cell density (30 00&50 000 cells/ml) was measured with a Model Z Couher Counter fitted with a 200 hum aperture tube. The temperature-pressure apparatus was patterned after one originally designed by Marsland [12] (cf [31]). Pressure up to 14 000 psi was attained at the rate of 5 000 usiistrokelsec. Various magnitudes of pressure, 5 OO&ldOOO psi (equivalent t;o 344.5 ): lo5 - 689 x IO6 N/m2 or Pascals) were aaolied for durations of l-4 min; the pressure was released virtually instantaneously by means of a needle value. The cells were transferred to flasks and placed into a shaker bath at 28°C. It required 2 min to disassemble the pressure apparatus. The pressure pulse treatment was repeated 5-7 times. A standardized recovery period of 30 min was maintained between pressure pulses. The end of the last pressure treatment was designated EP. Ceil samples (at 28°C) were removed from the flasks at various times after EP and the ‘division index’ (i.e. the % cells showing cytoplasmic furrows at any given time) and cell density were determined. The division maximum, occurring at a time when the majority of cells displayed cytoplasmic furrowing, was determined by plotting the changes in the division index as a function of time.
Biochemical procedures In the isotope centrifuged and prior to the last were added to Exptl
incorporation studies the cells were resuspended in inorganic medium [3] pressure pulse. At 2 min EP, the cells a flask containing one of the radio-
Cell Res 90 (1975)
active precursors (final concentrations: 0.3 pCi/ml W-thymidine, 2.5 &i/ml 3H-uridine, or 0.05 &i/ml “C-phenylalanine). At appropriate times, duplicate 0.5 ml aliquots were removed and immediately placed in an ice bath. An equal volume (0.5 ml) of cold trichloroacetic acid (TCA) was added to each aliquot (10 % TCA for DNA and protein determinations and 20 qb TCA for RNA determination). The aliquots were then allowed to remain at 0°C for 12 h. For phenylalanine incorporation studies, the samples were subjected to an additional procedure; the samples were placed in a bath of 90°C for 15 min. The radioactive material was collected on glass fiber filter pads (Reeve Angel, Clifton, N.J.) and washed with TCA, 95 and 100 % ethanol. The dried filter pads were placed in scintillation vials containing 15 ml of scintillation fluid (5.5 g of Permablend I (Packard Instrument Co. Inc., Downers Grove, Ill.) and toluene to make 1 liter). The radioactivity was measured with a Packard Tri-Carb liquid scintillation spectrometer. The counting efficiency was 33 “b for 3H and 85 :,, for ldC.
Both 5-3H-uridine (spec. act. 20.0 Ci/mM) and 2-Wthymidine (spec. act. 51.8 mCi/mM) were obtained from Schwarz BioResearch, Orangeburg, N.Y. L-3l*C-phenylalanine (spec. act. 15.3 mCi/mM) was purchased from Amersham/Searle Corporation, Arlington Heights, Ill., USA.
RESULTS Preliminary observations It has been previously reported that high pressureblocks cytokinesis and that moderate pressuresdelay division in heat synchronized Tetrahymena [9]. Log growth phase Tetrahymena are similarly affected by high hydrostatic pressure [ 111. The effects of high pressure on log cells vary in severity depending upon the magnitude of the pressure and the duration of the pressure treatment. High pressures (10 000 psi) for a long duration (10 min) drastically affect logarithmic growing cells causing them to become round-this is accompanied by a cessation of forward movement, although uncoordinated ciliary activity continues. A short time (5-10 min) after decompression most of the cells show some slow movement; they retain their roundish appearance and display prominent cytoplasmic vacuoles.
Pressure-induced
division synchron)!
121
Fig. I. Abscissa: duration of pressure (min); ordinate:max. % dividing. 0, 5 pressure pulses; m, 7 pressure pulses. Induction of division synchrony as influenced by duration of pressure, number of pressure pulses and magnitude of pressure. In all cases the cells were subjected to compression in PPL growth medium for a specified duration (l-4 min) and permitted 30 min for recovery between pulses in a shaker bath at 28°C. Under certain specified pressure-duration studies multiple division maxima were recorded, these are shown as irregular profiles at 7 500 psi for 3 and 4 min and at 10 000 psi for 1 min. Optimum synchrony was attained following 7 pulses at 7 500 psi for 2 min duration, and following 5 pulses at 8 500 psi for 2 min duration.
Lower magnitudes of pressure (7 500 and 8 500 psi) for short durations (l-4 min) did not induce major changes in the shape or activity of the cells. The posterior portion of some cells began to round 2 min after compression and this shapechange was more pronounced 4 min after compression. Ciliary velocity was also slightly reduced. Upon decompression the cells exhibited a rapid increase in movement; within 2 min the compressedcells displayed movement which was comparable to non-pressurized control cells. Previous studies [ 151 demonstrated that following pressure treatment abnormal patterns of nuclear morphology were observed when cells were allowed to remain in their culture flasks without agitation. The aberrations were attributed to 0, deprivation following the pressure treatment; nuclear abnormalities were not evident in pressurized cells that were subjected to gentle agitation following decompression.Thus the subsequent experiments were designedto employ moderate pressuresfor short durations and to in-
elude gentle agitation during the periods between pressuretreatments. Induction of synchrony From the foregoing observations it was evident that in order to induce synchrony it would be necessaryto usea pressure-duration combination which would produce a significant division delay but would not markedly affect cellular shape and activity. Since Scherbaum & Zeuthen [18] have established that a high degree of synchrony is induced following a series of cyclic temperature shocks at 34°C for a duration of 30 min followed by a recovery period of 30 min at 28°C a series of experiments were designed in which a duration of pressure treatment would be kept short and the recovery time would be kept constant (30 min). Various levels of hydrostatic pressure (5 000, 7 500, 8 500 and 10 000 psi) for different durations (l-4 min) were applied systematically 5 to 7 times. At both the low (5 000 psi) and the high (10 000 psi) pressuresit was not Exptl
Cell
Rrs 90 (1975)
122 Zimmerman
and Laurence
Fig. 2. A series of photomicrographs of Tefrahymena pyriformis prior to and after pressure induced synchrony. Division synchrony was induced by subjecting log growth cells (at 28°C) to a series of 5 pressure pulses, (8 500 psi for 2 min duration); each pressure pulse was separated by 30 min at atmospheric pressure (at 28°C). (a) Log growth cells; (b) cells 11 min after the last pressure pulse; (c) Tetrahymena cells 80 min after the last pressure pulse; (d) most of the cells in the field at 85 min after the last pressure pulse have undergone division.
possible to induce synchronous division (fig. 1). At the 5 000 psi level, the cells continued to divide and the division indiceswere comparable to control cells at atmospheric pressure. Whereas, at the 10 000 psi level, the cells developed abnormal shapes and displayed irregular swimming activity. A treatment of 10 000 psi for 1 min applied 5 times resulted in small multiple bursts of synchronous division (18 y0 divided at 80 min after the last pressure pulse (EP), 14 % divided at 100 min EP, 10 % divided at 110 EP and 8 % divided at 120 min EP); in these experiments division Exptl
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index was employed as a criteria for synchrony. A relatively high degree of synchrony (approx. 50 %) was routinely attained at pressuresof 8 500 psi for 2 min (cf fig. 2) and at 7 500 psi for 2 min. The optimum synchrony was attained at 7 500 psi after a series of 7 pressure pulses, whereas a comparable synchrony was attained at 8 500 psi after 5 pressure pulses. Increasing the number of pressure pulses at 8 500 psi to 7 did not increase the division synchrony. The first division peak occurred at 65-70 min and
Pressure-induceddivision synchrony
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Cell counts taken during the experiments showed a doubling of cell population after the first synchronous division and again a further increase after the second division. Log growth-phase cells at atmospheric pressure were monitored as controls. The size and shape of the pressure-synchronized cells were two to three times as large as log growth phase Tetrahymena and appeared similar to heat synchronized cells.
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3. Abscissa: time after EP (min); ordinate: (left) ‘% dividing (O-O); (right) cells/ml x 1OW. The development of division maximum in pressure synchronized Tetruhymena. Cells in PPL growth medium were pulsed 5 times at 8 500 psi for 2 min duration; each pressure pulse was separated by 30 min at atmospheric pressure. First and 2nd division peaks occurred at 80 and 220 min, respectively, after the last pressure pulse. The cell densities of pressuretreated cells ( x . . . x ) and log growth cells (A--A) are shown. Fig.
75-80 min EP at 7 500 and 8 500 psi, respectively. The second division peak (approx. 30 %) occurred approx. 210-230 min after the last pressuretreatment at either pressurelevel (fig. 3).
{E-40
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The incorporation of 14C-thymidine, 3Huridine and r4C-phenylalanine into the TCAprecipitable material was employed as an index of DNA, RNA and protein synthesis. Cells were transferred to inorganic medium prior to the last pressure pulse and treated with one of the radioactive precursors immediately after the last pressure pulse (2 min EP). As early as 10 min after the last pulse of pressure incorporation of specific radioactive precursors was determined in the acid-insoluble fractions. Between 20 and 40 min EP there was approximately a 2-fold increase in accumulative incorporation of 14Cthymidine and 14C-phenylalanine; and approximately a 50% increase in 3H-uridine incorporation (fig. 4). It should be noted that
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time after EP (min); ordinate: cpm x lo+. The incorporation of W-thymidine, SH-uridine (cpm [ x lO-2] per 8 000 cells) and %-phenylalanine (cpm [ x lo-*] per 8 000 cells) into DNA, RNA and protein respectively. Division synchrony was induced by a series of 5 pressure pulses of 8 500 psi for 2 min duration. Tetruhymenu were continuously exposed to the appropriate radioisotope commencing immediately after the last pressure pulse and the incorporation into the TCA-insoluble material was determined at various intervals thereafter. The incorporation of W-thymidine into pressure treated (O-O) and heat-treated ( x --- x ) cells are compared. The cell density of pressure treated and heat treated cells following treatment was 10 000 cells/ml and 12 000 cells/ml respectively; duplicate samples of 0.5 ml aliquots were removed for radioisotope determination (see text for details). Fig. 4. Abscissa:
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although the time for maximum division in inorganic medium was consistently delayed by 25 min, the division maximum was not decreased. DISCUSSION The present study confirms the prediction originally proposed by Dr Erik Zeuthen in 1957, namely, hydrostatic pressure treatments could be substituted for temperature treatments as a method for inducing synchronous division in Tetrahymena. Short pulses of hydrostatic pressure systematically applied to cells induce log growth phase cultures to divide synchronously. The division schedule and macromolecular synthesis in pressure synchronized Tetrahymena are similar to those found in heat synchronized cells. The increase in cell density of pressure synchronized cells compares favourably with the cell density increase of heat synchronized cells. The mechanism by which high pressure induces division synchrony in Tetrahymena pyriformis is subject to speculation, however, the hypothesis proposed by Zeuthen (cf [26]) concerning the synthesis of ‘division-essential proteins’ remains most attractive and the present studies support the hypothesis that the synthesis of essential material is altered by the physical effects of high pressure. The division maximum of pressure synchronized cells was approx. 50 % compared to a division maximum of 80-85 % for heat synchronized cells. The schedule of the first division (75 to 80 min E.P.) which resulted from the repetitive pulses of 8 500 psi, was comparable to that found with heat synchronized cultures. However, at lower magnitude of pressure (7 500 psi) the first division peak occurred approx. 10 min earlier. In general the duration of the second division cycle (145 min) of pressure-induced cells was greater than that found in heat-synchronized cultures (100-110 min). It is of interest to Exptl
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speculate that the small peak frequently observed in pressure synchronized cells appearing approx. 30 min after the major synchronous division index peak is similar to that reported by Zeuthen [27] in heat synchronized cells and may be comparable to the early and late participants in synchronous division. Zeuthen [27] has proposed that heat induces synchronous division in Tetrahymena by a differential extension of the G2 phase. Although no direct evidence is available, the similarity of the pressure-synchronized cells to heat-synchronized cells suggests a similar mechanism may be involved. Recently Zeuthen [28] has reported repetitive synchrony in Tetrahymena following heat shocks spaced a normal cell generation apart (160 min). In this technique for synchrony, each heat shock is initiated at the time the population has almost passed through the S phase, thus the heat shocks affect early G2. The display of free running synchronous divisions yield favourable division synchrony and replication. Systematically applied pressure pulses induce a high degree of division synchrony, however, DNA synthesis during the period from the last pressure pulse to the first synchronous division is not synchronized. RNA, DNA and protein synthesis continues throughout this same period, a pattern also reported for heat synchronized Tetrahymena. Although autoradiographic studies are essential to establish precisely the cycle stages in pressure synchronized cells, some preliminary information can be obtained from analysis of total incorporation profiles. The duration of G 1 in the second division cycle of pressure synchronized cells (in inorganic media) is comparable to the Cl of heat synchronized cells (in inorganic media). The Gl was estimated by measuring the length of time during which there was no net increase in the incorporation of thymidine. The Gl interval
Pressure-induced accounts for approx. 29 “/b of the second division cycle. The mechanism by which pressure induces division synchrony is subject to speculation. Although it is not possible to ascribe division synchrony to any single event, several morphological and biochemical events are implicated in the induction of division synchrony in Tetrahymena. Hydrostatic pressure induces morphological alterations, affects nuclear activity, and differentiation of the oral structure [13, 15, 201. Analysis of fine structure indicates that pressure reversibly disrupts microtubular elements in TetrahJjmena [6] as well as labile structures in other cell systems [31, 331. Biochemical studies have shown that transcription and translation in Tetrahymena are sensitive to hydrostatic pressure. Although pressure reduces the protein synthesizing capacity of cells by disrupting the polysomes [4], ribosomes isolated from pressure treated cells are able to synthesize polyphenylalanine as effectively as ribosomes prepared from non-pressure treated cells [8]. DNA synthesis [15], RNA synthesis and protein synthesis [lo] are reduced in cells following compression. Reduction in the synthesis of ribosomal precursor RNA and heterogeneous high molecular weight RNA are also found following compression [25]. In general, the present study supports the hypothesis that the prerequisites for cell division consist of a definitive sequence of biochemical events coupled with the formation of structural elements. Moreover the differential sensitivity of ‘division-essential’ materials to hydrostatic pressure results in the induction of synchronous division.
This paper is dedicated to Dr Erik Zeuthen, Carlsberg Foundation Biological Institute, who predicted in 1957 the feasibility of employing hydrostatic pressure for induction of division synchrony. This investigation was supported by the National Research Council of Canada.
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REFERENCES 1. Cameron, I L & Jeter, J R, J protozool 17 (1970) 429. 2. Cameron, I L & Padilla, G M, Cell synchrony. Studies in biosynthetic regulation (ed I L Cameron & G M Padilla). Academic Press, New York (1966). 3. Hamburger, K & Zeuthen, E, Exptl cell res 13 (1957) 443. 4. Hermolin, J & Zimmerman, A M, Cytobios 3 (1969) 247. 5. Johnson, F H & Eyring, H, High pressure effects on cellular processes (ed A M Zimmerman) p. I. Academic Press, New York (1970). 6. Kennedy, J R & Zimmerman, A M, J cell biol 47 (1970) 568. 7. Landau, J V, High pressure effects on cellular processes (ed A M Zimmerman) p. 45. Academic Press, New York (1970). 8. Letts, P J & Zimmerman, A M, J protozool 17 (1970) 593. 9. Lowe-Jinde, L & Zimmerman, A M, J protozool 16 (1969) 226. IO. -Ibid 18 (1971) 20. 11. Macdonald, A G, Exptl cell res 47 (1967) 569. 12. Marsland, D, J cellular camp physiol 36 (1950) 205. 13. Moore, K C, J ultrastruct res 41 (1972) 499. 14. Morita, R Y & Becker, R R, High pressure effects on cellular processes (ed A M Zimmerman) p. 71. Academic Press, New York (1970). 15. Murakami, T H & Zimmerman, A M, Cytobios 7 (1973) 171. 16. Padilla, G M & Cameron, 1 L, J cell camp physiol 64 (1964) 303. 17. Padilla, G M, Whitson, G L & Cameron, I L, The cell cycle. Gene-enzyme interactions (ed G M Padilla, Cl L Whitson & 1 L Cameron). Academic Press, New York (1969). 18. Scherbaum, 0 H & Zeuthen, E, Exptl cell res 6 (1954) 221. 19. Sedgley, N N & Stone, G E, Exptl cell res 56 (1969) 174. 20. Simpson, R E & Williams, N E, J exptl zoo1 174 (1970) 85. 21. Suzuki, K & Taniguchi, Y, Symp sot exptl biol 26 (1972) 103. 22. Villadsen, 1 S & Zeuthen, E, Exptl cell res 61 (1970) 302. 23. Wolfe, J, Exptl cell res 77 (1973) 232. 24. Wunderlich, F & Peyk, D, Exptl cell res 57 (1969) 142. 25. Yuyama, S & Zimmerman, A M, Exptl cell res 71 (1972) 193. 26. Zeuthen, E, Synchrony in cell division and growth (ed E Zeuthen) p. 99. Interscience, New York (1964). 27. - Exptl cell res 61 (1970) 3 11. 28. - Ibid 68 (1971) 49. 29. - Cell cycle controls (ed G M Padilla, I L Cameron & A M Zimmerman) p. 1. Academic Press, New York (1974). 30. Zimmerman, A M, The cell cycle. Gene-enzyme interactions (ed G M Padilla, G H Whitson & I L Exptl
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Cameron) p. 203. (1969). 31. - Int rev cytol 30 32. Zimmerman, A M 38 (1964) 454. 33. Zimmerman, S B
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pressure effects on cellular processes (ed A M Zimmerman) p. 179. Academic Press, New York (1970). Received May 20, 1974 Revised version received July 12, 1974
