J. Cell Set. 43, 7s-9i (1980)Printed in Great Britain © Company of Biologists Limited ig8o

THE RELATIONSHIP BETWEEN THE

EXCESS-DELAY PHENOMENON AND

TEMPERATURE-SENSITIVE PERIODS IN

TETRAHYMENA THERMOPHILA

JOSEPH FRANKEL* JYM MOHLERf ANDANNE KOOPMANS FRANKELDepartment of Zoology, University of Iowa, Iowa City, Iowa 52242, U.S.A.

SUMMARYAlthough temperatures of 37-5 and 39 °C allow continuous and rapid exponential growth of

wild type Tetrahymena thermophila, sudden shifts up to these temperatures can bring aboutlong excess-delays of cell division with accompanying resorption of developing oral primordia.A characteristic parameter of this delay-phenomenon is the physiological transition point,before which delays are maximal and after which they are negligible. When measured at arestrictive temperature that does not induce excess delays (36 °C), the end of the temperature-sensitive period of the cell division arrest of mutant cdaAl precedes the physiological transitionpoint, that of cdaHl roughly coincides with it, while the entire temperature-sensitive period ofcdaC2 comes after the physiological transition point. When cdaAi cells are exposed to 375 °Cor above, the manifestations of temperature sensitivity are drastically affected: the estimate ofthe end of the temperature-sensitive period (the execution point) becomes spuriously late, andthe characteristic division arrest following heat shocks is not manifested. The differentialeffects of the higher restrictive temperatures on cdaHl are more subtle, whereas those on cdaCzare negligible. We conclude that the excess-delay phenomenon involves a set-back of gene-mediated processes occurring at specific stages of the cell cycle.

INTRODUCTION

As first observed by Thormar (1959), sudden exposure to a high-temperature shockcan bring about long delays of cell division in wild type cells of the ciliate Tetrahymenapyriformis sensu stricto (then known as strain GL). This 'excess-delay' phenomenon ischaracterized by a delay of division which exceeds the duration of the heat shock andis larger the later the time in the cell cycle at which the shock is given. It was originallydiscovered in Tetrahymena, but has more recently been observed in a wide variety ofother cell types, including the bacterium Eschericfua coli (Smith & Pardee, 1970), thefission yeast Schizosaccharomycespombe(Kxan)h0h&cZe\ithen, 1971 ;Polanshek, 1977),the syncytial slime mould Physarum polycephalum (Brewer & Rusch, 1968; Wille,Scheffey & Kauffrnann, 1977; Tyson & Sachsenmaier, 1978), mouse L cells(Miyamoto, Rasmussen & Zeuthen, 1973), and eggs of sea urchins (Geilenkirchen,

This paper is dedicated to the memory of Professor Erik Zeuthen.• Author to whom reprint requests should be sent.f Present address: Room 16-720, Department of Biology, Massachusetts Institute of

Biology, Cambridge, Massachusetts 02139, U.S.A.6-a

y6 J. Frankel, J. Mohler and A. K. Frankel

1964) and of snails (Geilenkirchen, 1966). Mitchison (1971, chapter 10) has discussedits general significance at some length.

There are 3 aspects of this excess delay phenomenon that are especially important.One is that this phenomenon may be manifested not only following brief exposures tosublethal temperatures, as in the studies cited above, but also after sudden shifts totemperatures that are within the physiological range, demonstrated for Physarum byWright & Tollon (1978). A second, and crucial, aspect of the excess delay phenomenonis that it is a reflexion of a reversal of cellular development. This set-back hypothesisof the basis of excess delay was first proposed by Zeuthen (1958) for Tetrahymenapyriformis, and was confirmed, with respect to oral development, by Frankel (196211964, 1967) and Williams (1964&), who observed resorption of advanced oral primor-dia of T. pyriformis following heat shocks. A third important aspect of this phenomenonis that, in Tetrahymena at least, there is a sudden transition from maximal to no excess-delay response near the onset of cell division (Thormar, 1959), with a concomitantstabilization of oral development (Frankel, 1962) that also approximately coincideswith the onset of cell division (Williams, 1964a). This is the 'physiological transitionpoint' of Rasmussen & Zeuthen (1962), to which corresponds the morphogenetic'stabilization point' of Frankel (1962).

The temperature-sensitive periods of mutations at 3 loci, at which lesions bringabout arrest at specific stages of cell division in Tetrahymena thermophila, have beendetermined at 36 °C, a temperature which is restrictive for the 3 mutations but whichdoes not bring about excess delay of cell division (Frankel, Mohler & Frankel, 1980).The upper limit for continuous growth in this species is 40 to 41 °C (Nyberg, 1974;Schoephoerster & Frankel, unpublished observations). Previous work on generation ofexcess delays in T. thermophila has been carried out at sublethal temperatures in therange of 42 to 43 °C (Gavin, 1965; Suhr-Jessen, 1978). In this investigation, wedemonstrate that in standard peptone media typical excess delay patterns are generatedfollowing exposures to temperatures of 37-5 and 39 °C. We show here that the excessdelay phenomenon does interfere with the manifestation of temperature-sensitiveperiods that occur prior to the physiological transition point, and that the effects onthese temperature-sensitive periods provide a new kind of confirmation of the set-backhypothesis.

MATERIALS AND METHODS

Stocks and media

All cells used in this study were of inbred strain B of Tetrahymena thermophila. Three mutantstrains, homozygous for each of 3 cda (cell-division-arrest) mutant genes, cdaAl, cdaC2 andcdaHl, and one wild type strain (B-1975, II) were used in this study. The characteristics ofthese strains are described in the accompanying paper (Frankel et al. 1980).

The culture medium used for maintenance of stocks and also for single-cell culture experi-ments and most mass-culture experiments contained 1 % proteose peptone and o-i % DifcoBacto yeast extract (i % PPY). The only exceptions were a few experiments in which masscultures of cdaAi and cdaC2 cells were shifted to high temperature, in which a richer medium(2 % proteose peptone plus 0-5 % yeast extract) was used.

Excess-delay and TSPs of Tetrahymena mutations 77

Single-cell procedures

Single-cell techniques and protocols used were the same as described in the accompanyingpaper (Frankel et al. 1980).

Mass culture procedures

Batches of medium (150 ml) in 500-ml Fernbach flasks (Jena Glaswerk) were inoculatedwith about 30000 cells (200 cells/ml) from 1-day-old tube cultures and incubated at 28 CCwithout shaking or aeration for 16 to 18 h (5 to 6 generations). Experimental flasks were thentransferred to a waterbath set at temperatures ranging from 35-5 to 40-0 °C, depending on theexperiment. They were swirled for a few minutes after the transfer, to hasten attainment of thehigher temperature; the equilibrium temperature in the flask, 0-5 °C less than the average bathtemperature, was attained about 15 min after transfer to the bath. Following equilibration, theflasks were maintained in the bath without shaking or aeration. In some experiments, theflasks were kept at the high temperature for the remainder of the experiment. In others, theywere returned to the permissive temperature (28 CC) 30 min after the initial shift to the restric-tive temperature. The time required for re-equilibration to 28 °C was not precisely measured,but was probably about 10 min. Although nominally 30 min long, the actual duration of thehigh-temperature shock was closer to 20 min if reckoned in terms of time actually spent at therestrictive temperature.

In all mass-culture experiments, samples were taken at intervals starting 2 h before theinitial temperature shift and prepared for cell counts and silver staining. Cells were preparedfor counting as described earlier (Frankel, 1965) and counted in a model A Coulter Counter(Coulter Electronics). Silver staining and scoring of slides was carried out as described earlier(Frankel et al. 1980).

Data analysis

Execution points were defined as the time at which 50 % of the cells became able to dividefollowing a shift to restrictive temperature. To obtain a measure of the execution point as aproportion of the cell cycle, these times were compared to the interpolated median generationtime, as explained in the accompanying paper (Frankel et al. 1980). Standard parametric andnonparametric statistical methods were used, as described by Sokal & Rohlf (1969).

RESULTS

Effects of high temperature treatment on the cell division cycle

The effects of continuous and brief exposures to different high temperatures oncell multiplication of mass cultures of wild type and cdaAi and cdaC2 mutant cellsare shown in Fig. 1. Population doubling times at 28 °C were about 3 h for all 3genotypes. Wild type cells (O) shifted from 28 to 35 °C (panel ID) manifested animmediate transition to a new, higher growth rate. A shift from 28 to 39 °C, however,brought about a total cessation of growth for 1-5 h, followed by a semisynchronousburst of division before the culture returned to rapid exponential growth (panel I A).Shifts from 28 to 36 or 37-5 °C resulted in intermediate responses (T. thermophila cangrow exponentially at temperatures up to 40 °C, and grows fastest at 35 °C). Effects ofbrief exposures to temperatures ranging from 36 to 39-5 °C were remarkably similarto those observed following simple shifts to the corresponding high temperatures(compare plots with solid lines in the left (I) and right (II) columns in Fig. 1). Hence,the effect of a sudden shift to a high physiological temperature can be thought of ascomprising a transient initial shock effect followed by an adjustment to the new

J. Frankel, J. Mohler and A. K. Frankel

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Excess-delay and TSPs of Tetrahymena mutations 79

conditions; the transient shock effect on wild type cells is nonexistent after transfer to35 °C, mild in the 36-37-5 °C range, and severe at 39 °C.

Cultures of cdaAi ( x ) and cdaC2 ( # ) mutant cells shifted to 36 or 39 °C underwentslight increases in cell number followed by a cessation of cell multiplication due tofission-arrest (Fig. 1, panels Ic, I A). The temperature-shock protocol, however,allows comparison of the transient physiological effect of high temperature on mutantand on wild type cells, since under these conditions only a relatively small proportionof the mutant cells suffer fission arrest. The effects of 36 and 39 °C shocks on mutantcells were similar to those on wild type cells, whereas at 37-5 °C the mutants were moreseverely affected than the wild type (Fig. 1, column II).

The physiological effects of temperature shifts on the cell cycle can be explored ingreater depth employing single cell methods. Representative results obtained withwild type, cdaAi, and cdaC2 cells given 20-min exposures to 36, 37-5, and 39 °C atknown times in the cell cycle are shown in Fig. 2. The generation time of wild typecells was not affected by a 36 °C shock, was slightly affected by a 37-5 °C shock, andwas severely affected by a 39 °C shock. The response to a 39 °C shock was the classicexcess-delay phenomenon originally described by Thormar (1959).

The mutant stocks responded similarly to the wild type to 36 and 39 °C shocks(Fig. 2). At 36 °C, a slight but statistically significant division delay was observed incdaC2 and in cdaHi (not shown), but not in cdaAi. Mutant cells were again moreseverely affected by a 37-5 °C shock than were wild type cells. A positive regression ofgeneration time on interdivision age of exposure, with a (linear) slope significantlygreater than zero, was observed in both cdaAi and cdaC2 cells at 37-5 °C as well as at39 °C, and in wild type cells at 39 °C only. cdaHi was not tested at 37-5 °C, butresponded similarly to the other stocks at 39 °C (data not shown). The ' transitionpoint,' operationally defined as the time when 50% of the cells have made thetransition from maximal to no excess delay (Rasmussen & Zeuthen, 1962), is near 0-7of the cell cycle at 37-5 °C and generally closer to o-8 at 39 °C, corresponding to a timesomewhat before (37-5 °C) or approximately at (39 °C) the onset of division furrowing.

The maximal excess delay observed just prior to the transition point has been shownto be associated with active resorption of primordia of new oral apparatuses that arein late predivision stages of differentiation at the time of the shock (Frankel, 1962,1964a, 1967; Williams, 19646; Gavin, 1965). Cells undergoing resorption of oralprimordia either do not form division furrows or develop transient early furrows.

Fig. 1. Increases in cell number in cultures of wild type (O), cdaAi ( x ), and cdaC2(#) cells of T. thermophila subjected to temperature shifts (left panels) and shocks(right panels). The abscissa indicates hours at 28 CC relative to the initial shift to hightemperature. The upward-pointing vertical arrows show the time of the shift to hightemperature, while the downward-pointing arrows in the right panels indicate thetime of return to 28 °C. The ordinates indicate cell number in thousands of cells perml. In one experiment ( • , panel ID), wild-type cells were maintained at constant28 °C. The 3 curves within each of the right panels represent parallel culture flasks ofdifferent genotypes run within the same experiment. Panel IA shows 3 separateexperiments, with the mutants maintained in 2 % PPY. In all other experiments cellswere grown in 1 % PPY.

8o J. Frankel, J. Mohler and A. K. Frankel

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Excess-delay and TSPs of Tetrahymena mutations 81

This regression syndrome was also observed in the present study, by analysis ofsilver-impregnated slides made from samples of mass cultures fixed at o-5-h intervalsduring and after the 30-min exposure to high temperature. Oral primordia undergoingmembranelle formation during late predivision stages (4 and 5) are known to besusceptible to total resorption of oral primordia (Frankel, 1962, 1967). In wild type,no stage 4 and 5 oral primordia were resorbed after the 36 °C shock, few after the37-5 °C shock, and virtually all after the 39 °C shock. Concomitantly, the proportionof cells continuing normal development immediately after the shock declined slightlywith exposure to 36 °C, moderately following exposure at 37-5 °C, and fell to zeroduring the 0-5 h immediately following a 39 °C shock. The cdaAi and cdaC2 mutantcells manifested similar responses, except that the developmental effect of the 37-5 °Cshock was more severe, with a substantial proportion of the stage 4-5 oral primordiaundergoing resorption (more than half in cdaAi) 537-5 °C is thus near the threshold foreliciting the excess-delay/resorption syndrome, while 39 °C brings about a massiveeffect of this type.

In addition to bringing about excess delays of cell division, heat shocks also resultedin long-lasting arrest of division in cells exposed at specific times (Fig. 2, boxedsymbols at top of panels). Arrest could be distinguished from delay by persistence ofthe division furrow, and by a corresponding persistence of the completed new oralapparatus located posterior to that furrow. The arrests elicited after shocks at all 3temperatures applied near the end of the cell cycle in cdaC2, and after 36 °C shocksapplied in the middle of the cell cycle in cdaAi, were of types specific to these mutants(see Frankel et al. 1980). However, at 39 °C a few wild type cells as well as a generallylarger proportion of cdaAi mutant cells underwent fission arrest if shocked just afterthe transition point (Fig. 2). Occasional arrested cells were also observed in silver-impregnated samples of wild type and cdaAi mass cultures shocked at 39 °C; such

Fig. 2. Generation rime of wild-type and mutant single cells exposed to 20-min high-temperature shocks preceded and followed by maintenance at 25 °C, and of simul-taneous controls maintained continuously at 25 °C. Within each panel, the abscissaindicates the interfission age in min at which the shock began, while the ordinategives the resultant generation time. The cells arrested in division are included in abox at the top of each panel, utilizing the same abscissa as the remaining data points,while continuous-25 °C controls are shown in a bracket (C) to the right of each panel,sharing the same ordinates as the remaining points. Each row of panels gives theresults of a single experiment conducted with a single restrictive temperature (top,360 °C; middle, 375 °C; bottom, 390 °C), while each column represents a differentgenotype (left, wild-type, A; centre, cdaAi, x ; right, cdad, # ) . The verticaldashed lines in the middle and bottom panels indicate the average times of transitionfrom maximal to no delay of cell division, while the diagonal solid lines are theleast-square best-fit linear regression lines computed for all pre-transition cells. For themutants, results of different experiments at 37'5 and 39 °C are homogeneous (exceptfor cdaAi at 39 °C), and the regression lines and transition times are calculated fromthe data set comprising all of the experiments at that temperature, not just the singleone for which points are plotted. For wild-type, the heterogeneity among experimentswas significant and substantial, and the regression lines are only for the experimentsshown (a significant positive regression was observed in 2 out of the 3 wild-typeexperiments conducted at 39 °C, and in o out of 3 at 375 °C).

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Excess-delay and TSPs of Tetrahymena mutations 83

cells manifested a unique ' unequal arrest' syndrome that is characterized by a com-plete and relatively undistorted fission zone, a persisting posterior oral apparatus, anda tendency for the posterior division product to exceed the anterior product in size.This syndrome was rare in wild type cells following 39 °C shocks, but was muchmore common after 40 °C shocks.

Dependence of apparent execution points on restrictive temperature

The effects of the excess-delay and unequal-arrest syndromes on assessment oftemperature-sensitive periods (TSPs) of cell division mutants can be ascertained bycomparing the apparent TSP of these mutants at restrictive temperatures (37-5 and39 °C) that can elicit excess-delay to the actual TSP found at temperatures (35 and36 °C) at which no excess-delay occurs. The effect of temperatures above 36 °C onthe measured execution point (end of the TSP) will be shown to depend crucially onwhether the true execution point, measured at 36 °C, comes before or after the excess-delay transition point.

cdaC2. In the accompanying paper (Frankel et al. 1980), the TSP of this mutantwas shown to extend from stage MB to ML during cytokinesis (see Fig. 5 A of thispaper for diagrams of division stages). This temperature-sensitive period occursentirely after the physiological transition point.

The division stage at which execution occurs in cdaC2 is only slightly later at 39than at 36 °C (Fig. 3 B), the difference amounting to about 3 minutes of developmentaltime. Further, the division stages susceptible to arrest following heat shocks weresimilar at all 3 temperatures (Fig. 5 B). The reduction in percentage of B-stage cellsundergoing arrest after a 39 °C shock (Fig. 5; cf. Table 1) might have been due toinfliction of excess delay on such cells, a phenomenon to be considered in greaterdetail in connexion with cdaAi, below. Finally, cdaC2 cells arrested in divisionmanifested the same elongated appearance at all 3 temperatures. Hence, in thismutant, the expression of gene-specific temperature sensitivity is virtually unaffectedby the higher restrictive temperatures employed.

cdaAi. This mutant, unlike cdaC2, has a TSP that is entirely prior to the physio-logical transition point (Frankel et al. 1980). The measurement of this TSP is pro-foundly affected by the restrictive temperature. The effects of shifts to restrictivetemperatures ranging from 35 to 39 °C are presented in Figs. 3 and 4. When measuredby transfer to 35 or 36 °C, temperatures below the threshold for eliciting excessdelays of cell division, the cdaAi execution point appears well before the onset of cell

Fig. 3. Percentage of cells dividing following a shift from 25 CC to various restrictivetemperatures (36 °C, A; 37'5 °C, V; 39 °C, < )̂ as a function of the stage of cyto-kinesis at the time of the initial temperature shift. The abscissa is plotted according tothe duration of each stage [see Frankel et al. (1980) for duration, of stages, andFig. s, panel A, for drawings]. The ordinate gives the percentage of cells that completedivision, A, cdaAi cells; B, cdaC2 cells; c, cdaHi cells. Open symbols (A, V, 0 )indicate that the data were obtained with the culture-dish technique, closed symbols(A, • ) the pipette technique, and half-closed symbols (A, <•) that data from the2 techniques were combined. Numbers of cells are indicated in parentheses at eachpoint. The 36 °C data are taken from Frankel et al. 1980.

J. Frankel, J. Mohler and A. K. Frankel

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Fig. 4. Percentage of cdaAi cells dividing following a shift from 25 °C to variousrestrictive temperatures (35 °C, O; 36 °C, • ; 37-5 °C, V ; 39 °C, • ) as a function oftime in the cell cycle at the initiation of exposure, compared to the cumulativedivision curve of control cells ( • ) maintained continuously at 25 °C. The abscissa isplotted according to the interfission age, while the ordinate represents the percentageof cells that complete division. The number of cells at each of the points is given inparentheses. A total of 6 experiments is represented. The 36 °C data are taken fromFrankel et al. 1980.

division, at interfission ages of 0-54 and 0-58 respectively (Fig. 4). In contrast, theapparent cdaAi execution point measured at 37-5 °C is very much later (Fig. 4), nearinterfission age o-8 and the beginning of the E stage of fission (Fig. 3 A). This is notfar from the excess-delay transition point. At 39 °C, the apparent execution point isstill later (Fig. 4), near the middle of the division furrowing (Fig. 3 A).

The response of cdaAi cells to heat shocks also differs at different restrictivetemperatures. If such cells are shocked at 36 °C during a susceptible interval between40 and 80 min after the previous division (prior to the onset of the next division)about 25% of the cells become arrested in division (Table 1). In contrast, cells given37'5 o r 39 °C shocks during this same pre-division interval did not become arrestedin the subsequent division (Table 1). Thus, when shocks were applied at this time,the higher restrictive temperatures had the lesser effect.

In seeking to explain this apparent paradox, we should note that very few cells given36 °C shocks underwent long excess delays of cell division, whereas at the highertemperatures heat shocks brought about excess delays of cell division in the greatmajority of cells exposed within the susceptible interval (Table 1). The triggering ofexcess delays thus appears to be associated with inability to bring about fission arrest.

Analysis of the morphogenetic effects of high-temperature shocks can help explainhow the generation of excess delays might protect cells from becoming arrested in celldivision. The TSP of cdaAi corresponds to the time when membranelles are dif-ferentiating within oral primordia (Frankel et al. 1980). While a 36 °C shock does notaffect continuation of this process, higher shock temperatures bring about resorption

Excess-delay and TSPs of Tetrahymena mutations

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Fig. 5. Percentage of cells dividing following a 20-min exposure (shock) at variousrestrictive temperatures (36 °C, A; 37-5 °C, V ; 39 °C, <» as a function of the stageof cytokinesis at the time of the initial temperature shift. The abscissa and ordinatesare plotted as in Fig. 3, and the appearance of normally dividing cells in each stage isindicated in panel A (cf. Materials and methods of Frankel et al. 1980). Note thebreak in the ordinates in panels A and c. A, cdaAi cells; B, cdaC2 cells; C, cdaHi cells.Numbers of cells are indicated in parentheses at each point. It should be noted that apaucity of 37s and 39 °C data in panel B makes accurate plotting of the respectivecurves impossible beyond the MB stage; however, the limited data available suggestthat these curves would resemble that plotted for 36 °C. The 36 °C data are taken fromFrankel et al. 1980.

Table 1. Temperature-dependence of responses to heat shocks during the interval of susceptibility to division arrest

Genotype ... cdaAi cdaCi cdaHiSusceptibleinterval,36 °C ... 4o'-8o' 6o'-i3o' 5o'-i2o'

Restrictivetemperature, % de- % ar- % de- % ar- % de- % ar-

°C layed* rested (n) layed* rested (n) layed* rested (n)360 10 25 (57) 6 39 (67) 25 30 (117)37-5 86 3 (74) 47 25 (60) — — —39-o 91 3 (64) 69 23 (62) 57 18 (65)

• The criterion for delay is a generation time of 150 min or more in cdaAi and cdaC2; 180 min or more in cdaHi. Cells in the delayedgroup can easily be distinguished from non-delayed cells at 37-5 and 39 °C, while at 36 °C the distinction is more arbitrary (see Fig. 2).

Excess-delay and TSPs of Tetrahymena mutations 87

of developing oral primordia. This resorption can be viewed as a visible manifestationof a set-back of cellular development (Zeuthen, 1958). In this view, cdaAi cells thatare exposed to 37-5 or 39 °C prior to the excess delay transition point are pushed backto an early phase of the cell cycle, prior to the cdaAi TSP. If they remain at the hightemperature, they eventually resume development, reach the TSP, and becomearrested in division. However, if they are instead returned to the permissive tempera-ture before they resume development, they will pass through the TSP at 25 °C anddivide.

If the above interpretation is correct, then cdaAi cells suffering excess delay as aresult of a 37-5 or 39 °C shock should, after they resume development, still be subjectto division arrest as a consequence of a second 36 °C shock. In order to test this, cellswere maintained in micro-drops on culture dishes and given a 20-min exposure to37-5 °C, returned to 25 °C, and then, after a suitable interval (calculated so as toplace as many cells as possible in the temperature-sensitive period), given a second20-min exposure to 36 °C. Nine out of 33, or 27% of the acdAi cells given the firstexposure at 37-5 °C during the interval of susceptibility to division arrest werearrested by the second exposure to 36 °C. This proportion is similar to the 25 %observed after a single 36 CC shock applied during the susceptible interval(Table 1).

The set-back interpretation explains why the apparent cdaAi execution point at37-5 °C is close to the excess-delay transition point: cdaAi cells cease to be susceptibleto fission arrest following the temperature shift at the same time that they cease to besusceptible to a set-back of development that forces them to re-traverse the TSP atthe restrictive temperature. This, however, does not fully account for the effect of39 °C, at which the cdaAi execution point is substantially later than the physiologicaltransition point (compare Figs. 2 and 4) and heat shocks can bring about fission-arrestin dividing cells (Fig. 5 A). The apparent reason for this discrepancy is that at 39 °C anew nonspecific effect is superimposed: as already noted (pp. 82-83), cdaAi cellsexposed to 39 °C during early cytokinesis, after the transition point, can be preventedfrom completing division with a morphological pattern of unequal arrest that isdissimilar from the specific cdaA arrest pattern observed following 36 °C shocks andsimilar to the type of division arrest observed in wild type cells occasionally after 39 °Cshocks, and commonly at still higher temperatures. We therefore believe that this latephase of susceptibility of cdaAi cells to division arrest induced at 39 °C is probablyunrelated to any specific malfunction of the cdaAi gene product.

We conclude that the true cdaAi execution point is that measured at 36 °C (and35 °C). The apparent execution point measured at 37-5 °C is later, due to the excess-delay phenomenon, whereas the very late apparent execution point at 39 °C reflectsthe nonspecific arrest of dividing cells together with the excess delay of pre-dividingcells.

cdaHi. The cdaHi execution point measured at 36 °C is intermediate between thatof cdaAi and cdaC2 (Frankel et al. 1980), and comes approximately at the start ofcytokinesis. When assayed according to division stage (Fig. 3 c) the cdaHi executionpoint was at the beginning of the E stage at 36 °C, but near the end of the E stage at

88 J. Frankel, J. Mohler and A. K. Frankel

39 °C. Since the excess-delay transition point (at 39 °C) is situated near the beginningof the E stage, the execution point measured at 36 °C is roughly coincident with thetransition point, but the execution point at 39 °C seems to fall somewhat later.

Turning to effects of 20-min heat shocks, the proportion of cdaHi cells arrested isonly slightly (and not significantly) lower at 39 than at 36 °C (Table 1). However,there was a clear difference in the stage of these cells at the beginning of the heat shock.Virtually all of the cdaHi cells that subsequently became arrested after a 36 °C shockwere in the B stage or earlier when the shock began, whereas most of the cells thatbecame arrested after a 39 °C shock were in the E stage at the onset of the shock(Fig. 5 c). This suggests that, as in the other mutants, prefission cdaHi cells that mightotherwise have undergone division arrest did not do so at 39 °C, probably because theywere set back and therefore could not develop into that arrest during the shock. Cellsexposed to 39 °C during the E stage could no longer be set back and therefore becamearrested in division.

The nature of the division arrest of E-stage cdaHi cells shocked at 39 °C was notapparent from the study of living cells; however, examination of silver-impregnatedslides of a cdaHi mass culture shifted to 39 °C indicated that the cells that becamearrested in division shortly after the shift did not show the relatively undistortedpattern of unequal arrest observed in cdaAi and occasionally in wild-type cells underthese conditions, but instead manifested a highly distorted pattern of fission zonedevelopment that is diagnostic of cdaHi (Frankel, unpublished). It is thereforepossible that the execution point of cdaHi manifests a true temperature-dependence,being slightly later at 39 than at 36 °C.

DISCUSSION

There are 2 effects of sudden shifts to high temperature that can cause a change inthe apparent execution point of the temperature-sensitive mutations. One of these isthe tendency toward a nonspecific fission-arrest, observed occasionally in wild-typecells and more frequently in cdaAi cells following a shift to 39 °C. Since 39 °C is nearthe upper extreme of the normal physiological range, and this type of fission-arrest iscommon in wild-type cells shifted to higher temperatures outside of the physiologicalrange, it is likely that the enhanced susceptibility of cdaAi cells to this type of arrestis the result of some relatively nonspecific side-effect of the cdaAi lesion. Thiscomplication does not occur at 37-5 °C or less.

The other, more interesting effect is associated with excess delays of cell division.The results presented here support the conclusion that this excess delay phenomenonis a reflexion of a true set-back in the developmental sequence leading to cell division.This is demonstrated especially clearly with cdaAi, whose entire temperature-sensitive period (TSP), as measured at 36 °C (Frankel et al. 1980), occurs prior to thephysiological transition point. When cdaAi cells that have passed the execution pointare shifted to 37-5 °C, they become arrested in division, with the same morphologicalabnormality that is seen in pre-execution cells shifted to 36 °C. The most reasonable

Excess-delay and TSPs of Tetrahymena mutations 89

inference is that these cells have first been set-back in the cell cycle and later re-traverse the cdaAi TSP at the restrictive temperature, and thus become arrested.However, when such cells are given a 20-min shock at 37-5 °C, they do not becomearrested, presumably because they are set back to a point in the cell cycle prior to theTSP, and then re-traverse that period after the end of the shock, when they are againat the permissive temperature. That they are actually prior to the TSP following the37-5 °C shock is indicated by the fact that such cells are now susceptible to a second(36 °C) shock.

The above-described features have important implications for the determination ofgene-specific execution points of temperature-sensitive (TS) mutations. An executionpoint is clearly suspect if it is obtained at a temperature at which excess delays can beelicited and if it coincides with the physiological transition point. If high-temperatureeffects on cdaAi had been measured only at 37-5 °C without taking the consequencesof the excess-delay phenomenon into account, a spurious estimate of its executionpoint would have been made. Wright & Tollon (1978), though working only withwild-type cells, made a comparable point for the myxomycete Physarum polycephalum.They observed excess delays of mitosis following shift-up to temperatures of 31 °Cand above, and pointed out that for this reason the restrictive temperature for analysisof TS mutations in that organism should not exceed 30 °C.

In addition to yielding false estimates of execution points, the set-back phenomenonmay mask a specific TSP, if this is assessed by heat shocks. A phenomenon identical inprinciple was observed by Lindsley & Poodry (1977) in their recent study of thetemperature-sensitive mutant Shibire in Drosophila melanogaster: if heat shocks weregiven at a temperature high enough to arrest development, then the temperature-sensitive developmental effects of the mutation were not manifested, although thebehavioural effects appeared in full force. In both Drosophila and Tetrahymena, theapparent paradox of a less severe development effect following exposure to a moreextreme temperature is resolved when we recognize that expression of the develop-mental effect of a TS mutant is contingent on development actually occurring.

The employment of a restrictive temperature at which excess delays of cell divisionare manifested does not automatically cast doubt on all TSPs measured at thattemperature. If the TSP is wholly after the physiological transition point, as is thecase with cdaC2, it will obviously not be affected by excess delays.

More interestingly, the excess-delay phenomenon may not affect all aspects of thecell cycle in the same way. Cell cycles have generally been shown to involve 2 or moreparallel sequences of events (e.g. Hartwell, Culotti, Pringle & Reid, 1974; Frankel,Jenkins & DeBault, 1976; Zeuthen, 1978; Fantes & Nurse, 1978; Soil, Stasi &Bedell, 1978). A temperature shift might set one sequence back, yet leave anotherunaffected. For example, macronuclear DNA synthesis in Tetrahymena is not setback by heat shocks that severely affect progress toward cell division (Jeffery, Stuart &Frankel, 1970; Frankel et al. 1976; Zeuthen, 1978). Assessment of the TSP of amutant affecting such a parallel process might not be perturbed at temperatures thatbring about excess-delays of cell division.

There may, however, be another more subtle type of parallelism that pertains even7 C K L 4 3

90 J. Frankel, J. Mohler and A. K. Frankel

to processes that are subject to set-back. Since the physiological transition point isoperationally recognized by a dramatic break in the nature of cellular responses toheat shocks and other environmental perturbations, such as inhibitors of cell respirationand of protein syntheses (Frankel, 1962; Rasmussen & Zeuthen, 1962), it is easy tothink of it as a major culmination point in a sequence of events leading to cell division.However, at least one process, that dependent on the cdaHi product, appears toproceed continuously across this apparent major discontinuity, as at 39 °C the cdaHiTSP begins well before, and ends shortly after, the physiological transition point.The special feature of the physiological transition point is therefore not any necessaryclose causal linkage to other events, but rather the termination of a period in whichnumerous preparations for division are subject to massive disorganization followingenvironmental perturbations. Some highly labile process is taking place during thisperiod that is intimately involved both in oral development and in preparation fordivision. The nature of this process is unknown, but its relationship to other eventsmay be considered as analogous to the building and glueing together of a house of cards,while the physiological transition point is analogous to the drying of the glue.

The authors would like to thank Dr George Schoephoerster for conducting preliminaryexperiments on the effects of different temperatures on population growth, Dr J. DawsonMohler for suggesting the 375-36 °C double shock experiment, and Dr Joseph P. Hegmann foradvice on statistical analysis. Valuable suggestions for improvement of the manuscript wereprovided by Drs Michael C. Newlon and Karl Aufderheide. This research was supported bygrant no. HD-08485 from the U.S. National Institutes of Health.

REFERENCESBREWER, E. N. & RUSCH, H. P. (1968). Effect of elevated temperature shocks on mitosis and on

the initiation of DNA replication in Physarum polycephahim. Expl Cell Res. 49, 79-86.FANTES, P. A. & NURSE, P. (1978). Control of the timing of cell division in fission yeast. Expl

Cell Res. 115, 3I7-329-FRANKEL, J. (1962). The effects of heat, cold and p-fluorophenylalanine on morphogenesis in

synchronized Tetrahymena pyriformis GL. C.r. Trav. Lab. Carhberg 33, 1-32.FRANKEL, J. (1964). Cortical morphogenesis and synchronization in Tetrahymena pyriformis

GL. Expl Cell Res. 35, 349-362.FRANKEL, J. (1965). The effect of nucleic acid antagonists on cell division and oral organelle

development in Tetrahymena pryiformis. J. exp. Zool. 159, 113-148.FRANKEL, J. (1967). Studies on the maintenance of development in Tetrahymena pyriformis

GL-C. I. An analysis of the mechanism of resorption of developing oral structures. J. exp.Zool. 164, 435-460.

FRANKEL, J. JENKINS, L. M. & DEBAULT, L. E. (1976). Causal relations among cell cycleprocesses in Tetrahymena pyriformis: An analysis employing temperature sensitive mutants.J. Cell Biol. 71, 242-260.

FRANKEL, J., MOHLER, J. & FRANKEL, A. K. (1980). Temperature-sensitive periods of muta-tions affecting cell division in Tetrahymena thermophila. J. Cell Sci. 43, 59—74.

GAVIN, R. H. (1965). The effects of heat and cold on cellular development in synchronizedTetrahymena pyriformis WH-6. J. Protozool. 12, 307-318.

GEILENKIRCHEN, W. L. M. (1964). The cleavage schedule and the development of Arbacia eggsinfluenced by heat shocks. Biol. Bull. mar. biol. Lab., Woods Hole 127, 370.

GEILENKIRCHEN, W. L. M. (1966). Cell division and morphogenesis in Limnaea eggs aftertreatment with heat pulses at successive stages in early division cycles. J. Embryol. exp. Morph.16, 321-327.

Excess-delay and TSPs of Tetrahymena mutations 91

HARTWELL, L. H., CULOTTI, J., PRINGLE, J. R. & REID, B. J. (1974). Genetic control of the celldivision cycle in yeast. Science, N.Y. 183, 45-51.

JEFFERY, W. R., STUART, K. D. & FRANKEL, J. (1970). The relationship between deoxyribo-nucleic acid replication and cell division in heat-synchronized Tetrahymena. J. Cell Biol. 46,533-543-

KRAMHBFT, B. & ZEUTHEN, E. (1971). Synchronization of cell divisions in the fission yeast,Schizosaccharomyces pombe, using heat shocks. C.r. Trav. Lab. Carhberg 38, 351-368.

LINDSLEY, D. E. & POODRY, C. A. (1977). A reversible temperature-induced developmentalarrest in Drosophila. Devi Biol. 56, 213-218.

MITCHISON, J. M. (1971). The Biology of the Cell Cycle. Cambridge: Cambridge UniversityPress.

MIYAMOTO, H., RASMUSSEN, L. & ZEUTHEN, E. (1973). Studies on the effect of temperatureshocks on preparation for cell division in mouse fibroblast lines (L cells). J. Cell Sri. 13,889-900.

NYBERG, D. (1974). Breeding systems and resistence to environmental stress in ciliates.Evolution 28, 367-380.

POLANSHEK, M. M. (1977). Effects of heat shock and cycloheximide on growth and division ofthe fission yeast, Schizosaccharomyces pombe. J. Cell Sri. 23, 1-23.

RASMUSSEN, L. & ZEUTHEN, E. (1962). Cell division and protein synthesis in Tetrahymena, asstudied with p-fluorophenylalanine. C.r. Trav. Lab. Carhberg 32, 333-358.

SMITH, H. S. & PARDEE, A. B. (1970). Accumulation of a protein required for division duringthe cell cycle of Escherichia coii. J. Bact. 101, 901-909.

SOKAL, R. R. & ROHLF, F. J. (1969). Biometry. San Francisco: Freeman.SOLL, D. R., STASI, M. & BEDELL, G. (1978). The regulation of nuclear migation and division

during pseudo-mycelium outgrowth in the dimorphic yeast Candida albicans. Expl Cell Res.116, 207-215.

SUHK-JESSEN, P. B. (1978). Heat synchronization of cell division in Tetrahymena thermophilaand a mutant, NPi, with a temperature-sensitive defect for oral development. Carhberg Res.Comrnun. 43, 255-263.

THORMAR, H. (1959). Delayed division in Tetrahymena pyriformis induced by temperaturechanges. C.r. Trav. Lab. Carhberg 31, 207-226.

TYSON, J. & SACHSENMAIER, W. (1978). Is nuclear division in Physarum controlled by a con-tinuous limit cycle oscillator?^, theor. Biol. 73, 723-738.

WILLE, J. J., SCHEFFEY, C. & KAUFFMAN, S. A. (1977). Novel behaviour of the mitotic clock inPhysarum. J. Cell Sri. 27, 91-104.

WILLIAMS, N. E. (1964a). Induced division synchrony in Tetrahymena vorax. J. Protozool. 11,230-236.

WILLIAMS, N. E. (19646). Relations between temperature sensitivity and morphogenesis inTetrahymena pyriformis GL. J. Protozool. 11, 566-572.

WRIGHT, M. & TOLLON, Y. (1978). Heat sensitive factor necessary for mitosis onset in Physarumpolycephahim. Molec. gen. Genet. 163, 91-99.

ZEUTHEN, E. (1958). Artificial and induced periodicity in living cells. Adv. biol. med. Physics 6,37-73-

ZEUTHEN, E. (1978). Induced reversal of the order of cell division and DNA replication inTetrahymena. Expl Cell Res. 116, 39-46.

(Received 18 June 1979 - Revised 23 November 1979)

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