Effect of heat shock on growth and division of Stylonychia mytilus

Department of Zoology, University of Delhi, Delhi 110 007, India

Received February 21, 1990

KAUL, S. C. 1991. Effect of heat shock on growth and division of Stylonychia mytilus. Biochem. Cell Biol. 69: 23-28.

Stylonychia mytilus cells grown at 23°C exhibit an immediate arrest at GI and S stages in the cell cycle when subjected to a heat shock of 1 h at 35°C. The duration of arrest was seen to be dependent on the stage at which heat shock was given. It varied from 3 to 7 h and was synchronously accompanied by the delay in the completion of cell cycle. G2 and the early dividing stage Dl were found to be even more sensitive to heat shock than GI and S phases. Cells divide normally when heat shock was given at the late dividing stage D,. However, the GI stage of progeny cells was prolonged to 30 h from normal 5.5 h. These observations have been compiled from the cytological studies of normal and heat-shocked Stylonychia mytilus cells at different stages of cell cycle.

Key words: Stylonychia mytilus, heat shock, cell cycle, division delay.

KAUL, S. C. 1991. Effect of heat shock on growth and division of Stylonychia mytilus. Biochem. Cell Biol. 69 : 23-28.

Dans les cellules de Stylonychia mytilus croissant a 23"C, les stades GI et S du cycle cellulaire s'arrCtent immtdiate- ment lorsqu'elles sont soumises a un choc thermique d'une heure a 35°C. La durCe de I'arrCt depend du stade oh le choc est donne. Elle varie de 3 a 7 h et elle s'accompagne de facon synchrone du retard dans l'achtvement du cycle cellulaire. Le stade G, et un stade de division precoce (Dl) sont encore plus sensibles au choc thermique que les stades GI et S. Les cellules se divisent normalement quand le choc thermique est donne a un stade tardif de la division (D,). Cependant, le stade G, des cellules descendantes est prolong6 a 30 h alors que normalement il est de 5,5 h. Ces obser- vations ont Cte compiltes a partir d'etudes cytologiques de cellules de Stylonychia mytilus normales et de cellules ayant subi un choc thermique a differents stades du cycle cellulaire.

Mots clks : Stylonychia mytilus, choc thermique, cycle cellulaire, retard de la division. [Traduit par la Rtdaction]

Introduction advanced division furrow. Newly separated daughter cells were

In response to a rapid and transitory increase in temper- ature, eukaryotic cells display major changes in their pattern of protein synthesis (Tanguay 1988). It has been observed that transcription of most of the previously active genes is diminished during heat shock, while previously processed mRNAs remain stable (Findly and Pederson 1981). Although there are numerous reports in the literature on the bio- chemical response of cells to heat shock (reviewed by Schlesinger et al. 1982), there is still little information on the effects of this stress on cellular components or on prop- erties such as growth and cell division. The present study deals with the response to heat of the hypotrichous ciliated protozoa Stylonychia mytilus during the cell cycle. In these cells, analysis of the cell cycle events was facilitated by a cytological marker in the macronucleus, the replication band (site of DNA synthesis) which traverses the length of the macronucleus during the S phase. The replication band, as a marker, allows precise assessment of arrest and progress of growth and fission cycle. I have also studied the differen- tial sensitivity of different cell cycle stages to heat shock.

Materials and methods Stylonychia mytilus cells were cultured on the prey organism

Chlorogonium elongatum (phytoflagellate) as described earlier Waul and Sapra 1986). Experiments were performed on cells taken from clonal cultures that were between 800 and 1000 fissions old in the course of experimental duration.

Cell synchronization Synchronous cells were obtained from log phase mass culture

by picking up with a micropipette approximately 150 cells with an

'Present address: Fermentation Research Institute, 1-1-3 Higashi. Tsukuba, Ibaraki 305, Japan.

regarded as the GI (0 h) cells. I~ the-ensuing fission cycle, these cells maintained excellent synchrony in the growth cycle and divided within a span of 15-20 min.

Heat shock selection The intensity of heat shock was selected so as to have at least

90% cell survival. The recovery parameter was the ability of cells to establish viable clones when transferred back to 23°C after the heat shock. In different experiments, 50 G, cells were taken in four replicate tubes. Cells were shocked by immersion in a water bath at 35°C (+OS°C) for 60 min. This heat shock period was found to be optimum, as shown by the ability of cells to establish clones when returned to normal temperatures.

Mode of heat shock administration Three hundred cells, distributed equally in six tubes, were

immersed in a 35°C water bath for 60 min. After this treatment, the tubes were immediately transferred to culture chambers set at 23°C. For every point of treatment, observations were recorded for a complete division cycle (calculated from the point of treatment and including the duration of treatment).

Heat shock treatment Experiments were devised to determine the consequences of a

pulse-heat shock on specific stages of growth and fission cycle of Stylonychia mytilus. Duration of the fission cycle (10.5 h) was divided into 12 points; successive points of treatment were not spaced at equal intervals, but selected to represent sufficiently each cell cycle stage. Accordingly, heat shock schedule was as follows. ( i ) GI cells were selected at five points, spaced successively at 60-min intervals. ( i i ) S phase cells were chosen at three points (S,, S,, and S,), representing the progressive states of DNA synthesis and cytologically identifiable by positions of replication bands in macronuclei. These stages were also spaced at approximately 60-min intervals, successively. (iii) G2 phase cells were selected at two points, viz., ( a ) G,, cells that had recently completed the macronuclear S phase with two macronuclei proceeding to fuse with each other, and ( b ) G,, phase cells in which two macronuclei

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BIOCHEM. CELL BIOL. VOL. 69. 1991

TABLE 1. Delay in cell division following a heat shock at GI and S phases of cell cycle of Stylonychia mytilus. Delay includes the heat shock duration

Time (h k SD) taken for division cycle

Age of cells Stage of (h) treatment After heat shock Control Delay (h & SD)

NOTE: Number of experiments = 4; sample size = 50 in each stage.

TABLE 2. Arrest durations of replication bands in the macronuclei of cells exposed to the heat shock (a) Determined by appearance of replication bands

Appearance of

Stage of RB (h)

Delay (h) Delay (h k SD) heat After in the onset in division

treatment Age (h) heat shock Control of S phase (Ref. Table 1)

GI cells 0 11.5 5.5 6.0 6.1 + 0.32 1 10.6 4.5 6.1 6.0 k 0.29 2 9.6 3.5 6.1 6.3k0.11 3 6.5 2.5 4.0 4.1 k0.19 4 5.0 1.5 3.5 3.5 k0.44 5 4.7 0.5 4.2 4.2 k 0.10

- -

(b) Determined by progression of replication bands

Approximate time taken (h) to proceed through stages

of the S phase Stage of Approximate delay Delay (h + SD)

heat After (h) in entering in division treatment Age (h) heat shock Control the next stage (Ref. Table 1)

NOTE: Number of experiments = 4; sample size = 15 in each reading; RB, replication band. Durations for detecting the appearance of RB and end of arrested movement of RB can not be measured exactly. However, the error margin does not exceed & 5 % , because samples were examined at close intenrals of 30 min for the appearance and further movement of RB.

had fused in a single mass. (iv) D phase cells were also selected at two points, viz., (a) Dl cells showing the first division of the fused macronucleus and with early signs of cytokinetic furrow, and (b) D, cells that showed the second division of the fused macronuclei and an advanced state of the cytokinetic furrow.

Growth measurements Changes in the dimensions of cells were used as the marker to

record the cellular growth during G,. Control and heat-shocked cells were fixed in 0.01% OsO,. Measurements were made in arbitrary units by an ocular micrometer (Leitz) and converted to metric units with the help of a stage micrometer.

Protargol staining Stylonychia mytilus cells showed morphogenetic changes in

the cortex during the cell cycle. To study the effect of heat shock on the appearance and differentiation of various primordia, the

protargol staining technique described by Jerka-Dziadosz and Frankel (1969) was used.

Results Effect of heat shock on GI cells

Cells exposed to heat shock generally exhibit a prolonged division cycle. The extent of this delay ranged from 6 h in early GI cells to 4 h in late GI stages (Table 1). Heat- shocked cells showed a delayed onset of the S phase, as marked by the appearance of the replication band in the macronucleus. In all instances, entry of cells into S phase was postponed by a period that was equal to the period of delay in completion of the division cycle. For instance, fol- lowing heat shock, 0-h-old cells showed a delayed entry into S phase by 6 h, which was equal to the period of delayed

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CONTROL

of the cell =

HEAT SHOCKED

FIG. 1 . Schematic representation of sequence of cytological events from an experiment where the cells were heat shocked at 0-h age in GI phase. Cells showed delay of 6 h for entry into the S phase. The duration of arrest was equal to the delay in completion of divi- sion. A similar response was exhibited by cells heat shocked at other stages of the GI phase (six points investigated from 1 to 6 h age).

FIG. 2. Representative data from one experiment where cells of 0-h age (GI) were heat shocked. Control cells (0) followed a linear growth pattern, whereas heat-shocked cells (a) growth was arrested instantly. Exactly similar response of growth arrest was exhibited by cells at other GI stages (1-5 h, cf. Table 1). Growth arrest in all cases was equal to the delay in the division. Sample size = 25; no. of experiments = 4.

division (Table 2a and Fig. 1). Similar data were obtained at each of the five time points chosen for heat shock treat- ment. From these data it is obvious that heat-shocked cells were instantly arrested at the point of treatment and pro- ceeded towards S phase following a variable delay. This observation was further corroborated by the fact that fol- lowing heat shock such cells also showed an immediate arrest

in growth. In contrast to the control G, cells, heat-shocked cells showed no enlargement in cell size up to the beginning of S phase (Fig. 2). The growth arrest period at each point of treatment was equivalent to the period of delayed divi- sion. Analysis of cytological preparations revealed that treated cells were instantly arrested at the stage where treat- ment was given.

Effect of heat shock on S phase cells Cells heat shocked at three different stages in the S phase

showed delays between 5 and 7 h in completing the division (Table 1). In each experiment, a few cells were stained to ascertain the movement of the replication band in macro- nuclei. Examination of heat-shocked cells during the delay period revealed that the location of replication band remained unchanged. In each substage of the S phase, the heat shock produced similar consequences, i.e., an imme- diate arrest of the S phase. In the S1 phase, the replication band remained arrested for 5 h and then progressed to complete S phase. In S2 and S3 cells, the replication band progression was arrested for 7 h. In all instances, the S phase arrest period was equal to the delay in completing the cell division (Table 2b). At the end of the arrest period, replica- tion bands traversed at the same rate as those in control cells.

Effect of heat shock on the G2 cells The effects of heat shock on G2 cells were entirely dif-

ferent from those on GI and S phase cells. It was rather lethal to the G2 cells. Comparatively early G2 (G2J cells were slightly more tolerant to heat shock than late G2 (G2B) cells. The latter never survived beyond 30 min of treatment. In fact, most cells died within 15 min of heat treatment (Table 3). Prior to lysing, cells rounded up and became totally inactive.

Effect of heat shock on cells in the D phase Early dividing (Dl phase) cells rounded up within 10 to

15 min and lysed within an hour of the heat shock period (Table 3). Late dividing cells (D2) were not affected, as they proceeded to complete the fission within the scheduled time.

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TABLE 3. Relative sensitivity of cells in G2 and D phases to heat shock exposure

Percent survival State of

treatment 15 min 30 min 45 min 60 min Remarks

G ~ A 100 60 20 20 Survivors die after 5-6 h

G 2 ~ 2 o o o Cells lyse within 15 min

DI 40 20 5 0 Cells survive the heat shock up to 30 min only

D2 98 90 90 90 Only daughter cells show delay response

Now: Number of experiments = 10; sample size = 10 in each case.

D2 cells completed division in 10-15 min, even while being under the influence of heat shock treatment. However, the daughter progeny of heat-shocked cells showed a significant delay of 25 f 1.17 h in completing the next division cycle. The delay was due to the protracted GI phase. Instead of the usual duration of 5.5 h of the GI phase, these cells required about 30.5 h in completing this phase. Growth measurements of treated cells showed that the cell size remained constant throughout the arrested period.

Effect of heat shock on the oral primordia In early S phase (S1), oral primordia appeared in the

form of clustered kinetosomes (Fig. 3a). As cells proceeded in the S2 phase, the primordium increased in size and underwent differentiation in the form of parallel alignment of membranelles (Fig. 3b). Simultaneously, primordia for other ventral and marginal cirri also appeared. By late S, oral ciliatures (adoral zones of membranelles and undulating membrane) were completely developed and other primordia also differentiated into individual cirri (Fig. 3c).

Heat shock treatment given at all stages of S phase resulted in the arrest of primordia development. The developmental status of cortical primordia remained unaltered as long as the S phase was arrested (as described above). Following the arrest period, when the progression of the replication band began, primordia also started dif- ferentiating almost simultaneously (Figs. 3a-3c).

Discussion Response of cell in G1 phase

It is generally accepted that the G1 phase is the preparatory period in which cells accumulate inducers of DNA replication and undergo growth (Pardee 1989). How- ever, whether the GI growth occurs in distinct steps regulated by specific genes or in a continuous phase governed by messages from DNA templates is not yet clear.

Stylonychia cells, like other eukaryotic cells in the course of GI phase, undergo linear growth. Calculations reveal that these cells achieve 25% increase in their size before entry into the S phase. A heat shock treatment immediately blocks the growth processes. However, the results clearly demon- strate that cells heat shocked at five randomly chosen points do not show any specific arrest points in GI phase, imply- ing that there is no stepwise regulation. Therefore, it seems that there are no specific arrest points of growth in Stylonychia mytilus, as has been reported in Saccharomyces cerevisiae (Singer and Johnston 1982) and the mammalian cell (Pardee et al. 1978).

Effect on S phase cells Heat shock resulted in arrest in the progression of the

replication band. However, the latter sustained its cytolog- ical appearance during the arrest period (5-6 h). Evanson and Prescott (1970) have reported that in a closely related species, Euplotes eurystomus, heat shock caused changes in DNA synthesis as indicated by a shift in the replication band.

The above-mentioned response to heat shock was equally noticeable in early, middle, or late S phase cells in Stylonychia. However, in prokaryotes, it was found that heat shock during the early S phase was more effective than in late S phase (Kim et al. 1968). This might be due to the fact that protein synthesis is essential for initiation of DNA synthesis, but not for its continuation in the late S phase.

The disruption in DNA synthesis is probably related to the cessation of synthesis of S phase specific proteins. Heat shock is known to alter the protein profile of cells (Bienz and Pelham 1987), which in turn affects the continuation of DNA replication.

Response of G2 and Dl (early dividing) cells A noteworthy feature of G2 cells in Stylonychia mytilus

was their distinctly different response to heat shock in com- parison with G1 and S stages. Until the extreme end of the S phase, cells were able to survive following the heat shock. However, within a matter of a few minutes (after the S phase was over), the shock intensity, which was otherwise non- lethal, became lethal. Such extreme heat sensitivity was noticeable clearly for 30 min, the duration in which two macronuclei fuse together and randomize the distribution of DNA molecules.

Considering the following reports from the literature, it seems that the behaviour of Stylonychia mytilus G2 cells was unique. Mammalian GI and S cells are reported to be more sensitive than G2 (Miyamoto et al. 1973). Saccharomyces cerevisiae shows extensive delay in division and morphological aberrations (Polanshek 1977), whereas Tetrahymena cells do not exhibit any significant changes at corresponding stages (Frankel et al. 1980).

Similar to my observations of G2 sensitivity to heat shock, some other instances of extreme heat shock sensitivity in the literature are Tetrahymena in starved condition (Hallberg et al. 1984) and early developmental stages of Drosophila (Graziosi et al. 1980), sea urchin (Roccheri et al. 1981), and mouse embryo (Miiller et al. 1985). A possible explanation for lethality has been provided by Hallberg et al. (1984) in starved Tetrahymena, wherein death was caused by the reduced rate of synthesis of heat shock proteins. Since

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FIG. 3. Photomicrographs of silver-stained Stylonychia cells revealing the cortical primordia. Cells heat shocked at three different developmental stages ( ( a ) early S phase; ( b ) middle S phase, and ( c ) late S phase) showed that primordial development and differentia- tion was arrested, but there was no regression. OP, oral primordia; AZM, adoral zone of membranelles; FVT, primordia (no. 1-5) for the frontoventral cirral complex.

nonsurviving cells did not show different mRNA meta- bolism, they concluded that death was preceded by selec- tive RNA degradation.

Effect on late dividing stages The consequences of heat shock on late dividing cells are

not apparent immediately, because heat-shocked cells pro- ceeded for division as controls do. In the next division cycle, however, daughter cells showed a delay of 26 h. The delay was due to blocked early G1 phase. Since the cell size remained constant throughout the arrested period, it appeared that the arrest was not due to the slow rate of G, progression, but was clearly due to the block at initial stages of G1. The effect can not be assigned to nutritional reasons, as cells were observed to ingest and digest food at the same rate as control.

Such a phenomenon can only be explained by the assump- tion that heat shock at late dividing stages interrupts the syn- thesis of some kind of growth factors, which although not immediately required, are prerequisite for the following stages where the effect is noticeable. This has also been observed by Leof et al. (1982) in mammalian cells.

point of arrest. The delay in the cell cycle thus appears to be due to temporary freezing of the primordia development at the time when the heat shock is applied. The duration of delay in division was equal to the state of freezing of the cell cycle when heat shock was applied. Thus the response of two ciliate species is of a different nature with regards to the development of cortical primordia.

Division delay phenomenon Zeuthen (1974) has reported the occurrence of division

protein in Tetrahymena that along with many other factors prepares the cell for division. It is possible that heat shock affects such preparative assembly and thus results in the divi- sion delay.

Acknowledgements The author is thankful to the University Grants Commis-

sion (New Delhi) for financial assistance and the Science and Technology Agency of Japan for the present award of a Postdoctoral Fellowship. The help of Dr. G.R. Sapra and Dr. Y. Komatsu is duly acknowledged.

Effect of heat shock on oral primordia Tetrahymena cells when exposed to heat shock show corn- BIENZ, M., and PELHAM, H.R.B. 1987. ~echanisms of heat

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28 BIOCHEM. CELL BIOL. VOL. 69, 1991

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