Studies of membrane formation in Tetrahymena pyriformis

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of

1973 by Academic Press, Inc. reproduction in any .form reserurd

Experimental Cell Research16 (1973) 263-272

STUDIES OF MEMBRANE TETRAHYMENA

FORMATION

IN

PYRIFORMIS

VI. Variations in the Rate of Lipid Synthesis During Division of Heat-Synchronized Cells L. C. BAUGH and G. A. THOMPSON, JR Department of Botany,

University

of Texas, Austin, Tex. 78712, USA

SUMMARY Cells of Tetruhymena pyriformis, strain GL, induced to divide synchronously by alternating 30 min periods at 34” and 28”C, exhibited an increased rate of phospholipid synthesis during the initial stage of cytokinesis. This sharp peak of biosynthesis was evident from labeling studies with either 32P-orthophosphate or W-acetate. However, the period of enhanced lipid synthesis could be dissociated from cytokinesis by altering incubation conditions and was apparently associated more closely with recovery from the heat shocks than with the division process. Cells synchronized by a different procedure involving a single 30 min heat shock per generation showed no peak of labeling with 32Pi but retained the peak when tested with W-acetate. It appears that this latter perturbation was triggered by cytokinesis.

For some years studies of cells dividing in synchrony have provided a mechanism through which biochemical parameters could be correlated with specific events of the cell cycle [l]. Information obtained in this fashion has led us to realize that the synthesis of RNA, DNA, and a number of enzymes does not proceed at a constant rate throughout the cycle but rather takes place primarily during specific periods [2, 31. Because of the profound structura1 changes occurring prior to and during cytokinesis, many investigators have sought evidence for periodic variations in the rate of membrane production. This has indeed been indicated in some experimental systems. For example, different rates of lipid synthesis have been correlated with certain stages in the division cycles of various cell types grown in tissue

culture [4-71. The peak times of phospholipid synthesis were not the same in the different cell analysed, being sometimes in the S period [4, 51 and in other cases during or near cytokinesis [6, 7, 81. On the other hand, heat synchronized cultures of Tetrahymenapyriformis have been reported to synthesize lipids at a relatively linear rate throughout the recovery period and division process [9]. This finding, which contrasts with the results cited above, nevertheless carried considerable weight because of the superior degree of synchrony attainable with Tetrahymena and the large body of data correlating other facets of its metabolism. Our laboratory has been interested in the biogenesis of functionally different membrane systems found in Tetrahymena. In Exptl Cell Res 76 (1973)

264

L. C. Baugh & G. A. Thompson Jr

conjunction with our studies, we have reexamined this problem. In the previous report [9], certain periods in the cell cycle which interest us were not analysed. Furthermore, the methods utilized for lipid isolation were not designed to provide a quantitative recovery of phospholipids. Consequently, it seemed desirable to extend the earlier observations. We followed the same general approach used by the earlier workers [9], namely, that of measuring incorporation rates of radioactive lipid precursors at specific times in the cell cycle. However, in our study the labeling periods were selected because of their importante as times of structural change or of a division transition point (that point after which inhibitors no longer block or delay cytokinesis [l]). We also chose to measure rates of lipid synthesis over relatively shorter periods of time than were previously utilized. Employing these experimental modifications, we have observed a number of hitherto unreported properties of lipid metabolism.

MATERIALS

AND METHODS

of synchrony was almost as high with this procedure

as it was using alternating 30 min temperature shifts. Isotope administration Sample aliquots of 25 or 50 ml (100000 cells/ml) were labeled with 0.2 &i/ml Na ‘%-l-acetate (51 mCi/mmole; New England Nuclear Corp., Boston) or 1.6 &i/ml szP, (carrier-free, New England Nuclear) for exactly 10 min at several time intervals following the heat treatment. The end of the last heat shock (EH) was considered time zero for all subsequent periods. The times chosen for analysis were EH + 30, EH i- 50 (the physiological transition point, i.e., the time after which most inhibitors of protein synthesis have no capacity to delay division [l]), and 3 times during cytokinesis, EH-t 80 (the first visual indication of furrow formation, stage l), EH +90 (the stage at which the dividing cells are joined by only a narrow connection, stage 2), and EH -i-110 (the time 5 min following cell separation, stage 3). In some experiments additional times were analyzed.

Lipid and protein analysis Lipids were extracted directly from cells still in their medium by the method of Bligh & Dyer [15], followed by the removal of non-lipid contaminants by 2-3 (always 3 in 32Pi experiments) washes according to Folch et al. [16]. Radioactivity was measured in a Packard Model 3310 scintillation spectrometer. Analysis of lipid phosphorus and the distribution of radioactivity among the various lipid components was performed as described earlier [ll]. Rates of SH-4,5-leucine (29.8 Ci/mM, New England Nuclear) incorporation into protein were measured by the method of Byfield & Scherbaum [17] with slight modifications. Subcellular organelles were isolated as previously described [lo].

Culture conditions Experimental cultures of Tetruhymena pyriformis, strain GL, were grown to early logarithmic phase on a rotary shaker using a 500 ml Erlenmeyer flaskcontaining 200 ml enriched proteose peptone medium [13] and were synchronized according to the heat shock method of Zeuthen [l], which employs alternating 30 min periods of incubation at 34” and 28°C. Synchronous division occurs approx. 80 min after the last heat shock. The reproducibility of the data was dependent upon a high division index (9&95 %) and a close synchrony (70-80 %) of microscopically identifiable stages of furrowing of the dividing cell. This high degree of synchrony can best be realized if the culture is rapidly cooled to 28” following each heat shock. Figs 1 and 2 illustrate the high degree of synchrony achieved and identify the stages of cytokinesis we refer to below as stages 1 and 2. An alternative technique used for inducing synchronous division involved the application of a single 30 min 34°C heat shock per generation for 6 consecutive cell generations [14]. The period at 28” between heat shocks was 160 min. Following the last shock, division began in SO*3 minutes. The degree Exptl Cell Res 76 (1973)

RESULTS The initial series of experiments was designed to measure any changes in the rate of membrane lipid synthesis during or preceding cytokinesis. Fig. 3a compares the specific activities of phospholipids from whole cells incubated with 32Pi for 10 min periods immediately prior to EH and during the recovery period and 3 stages of cytokinesis. Even though some lipid synthesis continues throughout the heat treatment [9], we find that a return to the normal growth temperature at EH initiates an increase in the rate of phospholipid

synthesis.

The increase

con-

tinues until the cells regain the synthetic rate

Lipid synthesis in synchronized Tetrahymena cells

265

Fig. 1. Phase micrograph showing cells during the onset of cytokinesis. At this time, denoted stage 1, the first indentation of the division furrow becomes visible on one side of each cell. x 158. Fig. 2. Phase micrograph showing cells at stage 2 (see text for definition). x 165.

normally observed in 28°C cultures. From EH to EH +50 there is a slight increase in the rate of 32Pi incorporation, apparently representing this gradual recovery from the heat treatment. Following EH +50 the rate of phospholipid synthesis increases markedly, exhibiting a peak of activity at EH +80, the point in the cycle which coincides exactly with the first visual signs of cell furrowing. The rate of synthesis then declines through the subsequent stages of cytokinesis. The more gradual increase in the synthetic rate resumes following division (EH + 110). Examination of cells at longer times following EH showed that the synchrony was considerably poorer (division index 35-40~~) during the second division. Under these conditions we were unable to detect further significant changes in the rate of lipid synthesis. Also plotted in fig. 3 a are data establishing

the rate of 3H-leucine incorporation into protein during the period analysed. The incorporation of 3H-leucine proceeded in a linear fashion throughout the cell cycle as reported earlier by Crockett et al. [18]. We wondered if the apparent peak of phospholipid synthesis could be an artifact caused by variations in the cellular permeability to 32Pi. Earlier reports suggest that this is not the case; there is some periodic variation in 32Pi uptake, but the peak occurs well before cytokinesis [l]. Nevertheless, we repeated the experiments, using 14C-l-acetate, a molecule which differs greatly in physical properties from 32Pi but which is readily utilized by Tetrahymena for synthesis of the fatty acid components of phospholipids [I I]. Fig. 3 b illustrates that 14C-acetate incorporation follows the same basic pattern as found with 32P.1’ Exptl Cell Res 76 (1973)

266 L. C. Baugh h G. A. Thompson Jr

i

.. u l

1 1

a b

.

.

& .

.

/--

i

i

Figs 3. Abscissa: min after EH; ordinate: (left) a, b, phospholipid spec. act. (0-O) (rig& 3a) protein radioactivity n - w (cpm/ml culture).

%, of 28°C control;

(a) The incorporation of sZPi into phospholipids ( l - 0 ) and “H-leucine into protein ( n - n ) by aliquots of synchronized Tetrahymena cultures. Each point represents a single determination. The vertical dashed line denotes the beginning of cytokinesis (stage 1). All data reported in this and subsequent figures are from cultures containing 8-11 x lo4 cells/ml at EH. Points show beginning of 10 min isotope incubation with aliquots. (b) incorporation of W-acetate into phospholipids by aliquots of synchronized Tetvahymena cultures. Notations same as in fig. 3a.

The evidence reported in fig. 3 prompted us to speculate that increased lipid synthesis is required during the early stages of cell division. In order to test this possibility further, the following experiments were designed. The growth rate of Tetrahymena cultures is markedly dependent upon the oxygen supply available to the cells. By carrying out the heat shocks and subsequent incubation in a 2 500 ml wide form flask, the surface area and depth of medium were changed to 490 cm2 and 0.5 cm, respectively, as compared with figures of 64 cm2 and 3.8 cm for our standard 500 ml Erlenmeyer flask. Under these conditions, division occurs 18-20 min earlier than usual due to improved aeration. Labeling experiments with 32Pi and 14Cacetate (fig. 4) clearly indicated, to our surExptl Cell Res 76 (1973)

prise, that the peak of incorporation still occurred at almost the same time as found using standard conditions. Thin layer chromatographic analysis of the major lipids following the incubations described in fig. 4 with 14C-acetate or 32Piyielded the pattern shown in table 1. The distribution of radioactivity is not greatly different from that reported earlier with 14C-palmitate [l 11, and no significant variations could be detected at different times during the cell cycle. However, while short term incubations of this type are sensitive indicators of lipid synthetic patterns at the time, they do not reflect gradual alterations in lipid composition which may be occurring. For example, we were able to observe a slow increase in the tetrahymanol/phospholipid molar ratio from the normal level of 0.066 [19] to a peak of

Lipid synthesis in synchronized Tetrahymena cells

a

. .

.

267

b

. .

. . P

Fig. 4. Abscissa: min after EH; ordinate: phospholipid spec. act. (O-O) % of 28°C control. (a) Incorporation of azP into phospholipids by aliquots of highly aerated Tetruhymenu cultures. Notations same as in fig. 3~. (b) Incorporation of T-acetate into phospholipids by aliquots of highly aerated Tetrahymena cultures. Notations same as in fig. 3a.

approx. 0.087 during the heat shock treatment (table 1, column 6). After EH the ratio gradually declined to normal. A quantitative analysis of the individual phospholipids at various stages of the cell cycle has not been made, but visual inspection of thin layer chromatographs failed to disclose any obvious differences. A second modification in culture conditions was tested. Cells were grown and heat shocked in the wide form flasks as desrcibed above. However, in this case, a 485 ml/min flow of commercial tank nitrogen gas was introduced into the flask for a 15 min period starting at EH +25. As reported previously by Koch & Scherbaum [20], this hypoxic treatment produced a delay (approx. 25 min under our conditions) in reaching the 1st stage of division. The nitrogen treatment eliminated entirely the peak of 32Pi incorporation (fig. 5~). The peak of 14C-acetate in-

corporation was still observed at a somewhat reduced level and occurred at EH+70 (fig. 5b). The results of these latter two modifications strongly support the concept that the peak of lipid synthesis is associated not with the process of cytokinesis but rather with recovery from the heat treatment itself. The reduction or elimination of the peak following the nitrogen-induced delay may indicate that progress towards recovery from the heat shocks continues throughout the delay period. Thus it would seem that for our purposes the utility of the heat shock procedure for inducing synchrony is greatly reduced. It would hardly be feasible to study, as we wish to do, the subtle intracellular changes in lipid redistribution to various membranes if a large artifactual peak of biosynthetic activity is superimposed upon the normal processes. Other methods for inducing synchrony Exptl Cell Res 76 (1973)

268

L. C. Baugh & G. A. Thompson Jr

Table 1. Distribution of radioactivity with the indicated precursor

among major phospholipids after a 10 min incubation

% of total radioactivity in

Precursor

Time of sampling

H,=PO H,=PO: H,=PO H,=PO: W-acetate W-acetate W-acetate W-acetate

EH EH i- 80 min EH+llOmin 28” control EH EH + 80 min EH+llOmin 28” control

total phospholipid

PC

PE

AEPL

Tet./PL molar ratio

50 82 78 72

13 9 11 6 12 17 21 24

44 56 50 57 28 39 ::

20 24 23 22 15 9 9 10

0.089 0.071 0.067 0.067 0.087 0.069 0.067 0.066

Data derived from experiments presented in fig. 4. Percentages are averages of 3 expts and tetrahymanol data are averages of two determinations by the method of Thompson et al. [12]. PC, choline phosphatides; PE, ethanolamine phosphatides; AEPL, 2-aminoethyl phosphonolipids.

were considered. The use of hypoxic shocks has been successfully employed by some authors [21]. This method appeared to have some disadvantages for our work since the quantitative pattern of phospholipid labeling is upset in the absence of oxygen. Thin layer chromatographic analysis showed that cells incubated for 90 min under nitrogen divert into phosphatidylcholine over twice the % of fed 14C-palmitate that we previously reported [II] for aerobic cells. Even after air is reintroduced, the synthetic pattern remains abnormal in aliquots of cells tested at various times during the subsequent hour. On the other hand, the quantitative distribution of 32Pi in lipids is the same under anaerobic and aerobic conditions. The anomaly is presumably due to altered patterns of lipid desaturation. About the time these results were being observed, a new heat synchrony technique was introduced by Zeuthen [14]. The method calls for only one heat shock per generation, and the size and shape of the cells emerging from such a treatment are nearly normal. The pattern of lipid synthetic activity after Exptl Cell Res 76 (1973)

cells were synchronized using the new method is presented in fig. 6. Under these conditions there is no peak of radioisotope incorporation when 32Pi is involved. However, a peak corresponding to the 1st stage of division is seen when 14C-acetate is supplied. The distribution of radioactivity among the major phospholipids (table 2) was rather similar to that observed with the original Zeuthen procedure. Changes in the tetrahymanol/ phospholipid ratios were less pronounced using the new technique. Once again, the question arose-is the observed peak of 14C-acetate incorporation a part of the normal cell cycle or is it an artifact produced by the heat treatment? Unlike the first method tested, cells synchronized by the new Zeuthen procedure receive only one 30 min shock during the 270 min period preceding the beginning of the division analysed. It seems most unlikely that heatinduced artifactual fluctuations in lipid synthesis could be caused by any save the last heat shock. To test the effect of a single heat shock upon lipid metabolism, we carried out the experiment described in fig. 7. Clearly, a

Lipid synthesis in synchronized Tetrahymena cells

269

b

a

10

” :

Fig. 5. Abscissa: min after EH; ordinate:

phospholipid spec. act. (O-O) % of 28°C control. (a) Incorporation of 32PI into phospholipids by aliquots of highly aerated Tetrnhymena cultures flushed for 15 min (see bar above abscissa) with nitrogen gas. For experimental details see text. Notations same as in fig. 3a. (b) Incorporation of T-acetate into phospholipids by aliquots of highly aerated Tetrahymenu cultures flushed for 15 min (see bar above abscissa) with nitrogen gas. For experimental details see text. Notations same as in fig. 3a.

single 30 min heat shock given to cells in logarithmic growth does sharply depress fatty acid synthesis. However, recovery is a gradual process which is completed by 90 min without a detectable peak of 14C-incorporation. Thus the peak shown in fig. 6b would seem not to be effected by the terminal heat shock per se. DISCUSSION Little quantitative information is available regarding the membrane dynamics of dividing cells. As cytokinesis begins, it is likely that the rate of synthesis of certain specific membranes increases, perhaps at the expense of others. It would be of obvious importance to understand the factors responsible for redirecting structural proteins and lipids to new destinations, if such is indeed the case.

Because of the ease with which Tetrahymena can be induced to divide in a very precisely synchronized sequence of events, we judged it to be ideally suited for studying this phenomenon. Our previous work with Tetrahymena growing and dividing asynchronously has provided us with techniques for analysing the movement of newly formed lipids from the sites of their synthesis to several functionally different membranes within the cell [ll]. These experiments, repeated with synchronized cultures at selected intervals during the cell cycle, might provide quantitative data revealing changes in lipid deployment patterns. Our first step in this direction has been the above experiments measuring the rates of lipid synthesis by synchronized cultures. This knowledge is essential before detailed studies of subcellular fractions begins. Our choice of Exptl Cell Res 76 (1973)

210 L. C. Baugh & G. A. Thompson Jr . .

.

b

\f

Fig. 6. Abscissa: min after EH; ordinate: phospholipid (a) The incorporation of saPl into phospholipids the new procedure of Zeuthen [14]. (b) Incorporation hymena cultures synchronized by the new procedurelof

spec. act. (0 - 0) % of 28°C control. by aliquots of Tetrahymena cultures synchronized by of W-acetate into phospholipids by aliquots of TetruZeuthen [14]. Notations same as in fig. 3~.

a method for inducing synchrony was the classical method of repetitive heat shocks pioneered by Zeuthen [l]. Although this procedure yields cells which are grossly abnormal in size and lipid composition, it was our hope that lipid metabolism would return to normal during the 80 min that elapse between the terminal heat shock and the first synchronous division. The experiments described above indicate that lipid metabolism does not resume a normal pattern until after the first division has occurred. A sharp peak of phospholipid synthesis, at first thought to be an essential part of the division process, can be dissociated from that process by factors which hasten or delay division. The peak of synthesis seems more closely linked to EH, since it invariably

occurs at a fixed time following the last heat shock. Just why increased aeration or oxygen deprivation should not alter the time of this increased biosynthetic activity is not apparent. One assumes a ‘clock’ is set at EH and that the resumption of cell division and related processes then requires only a given period of restored metabolic activity. The nondependence of the pulse of increased lipid formation upon general metabolic activity suggests that it results from a specific time-dependent process which does not draw heavily on the cell’s energy supply. Whatever the reason for the observed peak of lipid synthesis, it is sufficiently pronounced to jeopardize the success of our intended studies on intracellular lipid redistribution.

Exptl Cell Res 76 (1973)

Lipid synthesis in synchronized Tetrahymena cells Table 2. Distribution of radioactivity with the indicated precursor

271

among major phospholipids after a 10 min incubation

% of total radioactivity in total phospholipid

Time of sampling EH EH+83 EH+113 28°C control EH EH+82 EH+112 28°C control

H,=PO H3=PO: H,=PO H,=PO: 14C-acetate W-acetate W-acetate W-acetate

53 88 70 73

PC

PE

AEPL

Tet./PL molar ratio

4 4

46 38 44 52 26

13 12 14 16 10 11 10 10

0.072’ 0.063’ 0.062’ 0.062a 0.079 0.058 0.062 0.064

2 18 30 26 27

zi 35

Data derived from experiments presented in fig. 6. Notations are as described in table 1. a The results of a single analysis

We consequently chose to seek a procedure causing fewer metabolic derangements. One such possibility, the use of hypoxic shocks, was tried and rejected. A second alternative,

an

10

40

60

10

100

110

110

Fig. 7. Abscissa: min after EH; ordinate: phospholipid spec. act. ( l -0) % of 28°C control. The incorporation of W-acetate into phospholipids by aliquots of Tetrohymena cultures exposed to a single 30 min heat shock. Notations same as in fig. 3a.

the modified heat shock procedure of Zeuthen [14], holds considerable promise. Using this latter technique, we were able to observe a high degree of closely synchronized division (division index > 85 ‘%) under conditions where no enhancement of 32Pi incorporation occurred. This indicates that a transient increase in the rate of phospholipid synthesis does not necessarily accompany cytokinesis. A peak of 14C-acetate incorporation is still found during the same early stages of division where it occurred using the original Zeuthen technique of more frequent heat shocks (fig. 4). However, our evidence indicates that in this case the enhancement in acetate utilization is indeed triggered by the division process. The tentative conclusion would be that increased fatty acid synthesis but not phospholipid synthesis is associated with cytokinesis. It will now be feasible to move ahead with greater confidence to the principal aim of the study, namely, charting the intracellular lipid redistribution during various periods of the cell cycle. The authors would like to express their gratitude to Dr Yoshinori Nozawa for his encouragement and Exptl Cell Res 76 (1973)

272 L. C. Baugh & G. A. ThompsonJr helpful discussions during the course of this work. We would also like to thank Stephen W. Hemperly for his technical assistance during the initial exneriments. The project was supported in part by NSF grants GB-16363 and GU-1598 and Robert A. Welch Foundation grant F-350. L. C. B. was the recipient of an award from the NSF Research Participation Program for College Teachers, and conducted a portion of the research at McLennan Community College, Waco, Texas.

REFERENCES 1. Zeuthen, E, Synchrony in cell division and growth (ed E Zeuthen) p. 1. Wiley. New York (1964). 2. Hanson, E D; Chemical zoology (ed M Florkin & B T Scheer) vol. 1, p. 395. Academic Press, New York (1967). 3. Mitchison, J M, Science 165 (1969) 657. 4. Bergeron, J J M, Warmsley, A M H & Pasternak, C A, Biochemj 119 (1970) 489. 5. Warmsley, A M H, Phillips, B & Pasternak, C A, Biochem j 120 (1970) 683. 6. Gerner. E W. Glick. M C & Warren. L._ J cell physioi 75 (1970) 275. 7. Bosmann, H B & Winston, R A, J cell biol 45 (1970) 23.

Exptl Cell Res 76 (1973)

8. Daniels, M J, Biochem j 115 (1969) 697. 9. Bvfield. J E. Henze. J & Scherbaum.I 0.I Life sci 6-(1967) 1099. ’ 10. Nozawa, Y & Thompson, G A, Jr, J cell biol 49 (1971) 712. 11. - Ibid 49 (1971) 722. 12. Thompson, G A, Jr, Bambery, R J & Nozawa, Y, Biochemistry 10 (1971) 4441. 13. Thompson, G A, Jr, Biochemistry 6 (1967) 2015. 14. Zeuthen, E, Exptl cell res 68 (1971) 49. 15. Bligh, E G & Dyer, W J, Can j biochem physiol 37 (1959) 911. 16. Folch, J, Lees, M & Sloane-Stanely, G H, J biol them 226 (1957) 497. 17. Byfield, J E & Scherbaum, 0 H, Anal biochem 17 (1966) 434. 18. Crockett, R L, Dunham, P B & Rasmussen, L, Compt rend trav lab Carlsberg 34 (1965) 451. 19. Thompson, G A, Jr, Bambery R J & Nozawa, Y, Biochim bioohvs acta 260 (1972) 630. 20. Koch, E G & Scherbaum, b H,Z allg Mikrobiol 7 (1967) 349. 21. Rooney, ..^ .-. .^D W & Eiler, J J, Exptl cell res 48 (lY67) 64Y.

Received June 6, 1972 Revised version received July 24, 1972