J. Cell Sci. 33, 1-23 (i977)
Printed in Great Britain
EFFECTS OF HEAT SHOCK AND
CYCLOHEXIMIDE ON GROWTH AND DIVISION
OF THE FISSION YEAST,
SCHIZOSACCHAROMYCES
POMBE
WITH AN APPENDIX
ESTIMATION OF DIVISION DELAY FOR S. POMBE
FROM CELL PLATE INDEX CURVES
MARK M.POLANSHEK*
Department of Zoology, University of Edinburgh, Edinburgh,
Scotland, EHg jjfT
SUMMARY
Effects of superoptimal temperature shock (HS) and the protein synthesis inhibitor, cycloheximide (CH), on growth and division of the fission yeast, Schizosaccharomyces pontbe, were
studied. Experiments on asynchronous cultures have shown that a 15-min HS of 41 °C inhibits
RNA and protein synthesis and growth in cell length while delaying mitosis, division, and
DNA synthesis. A 10-min CH pulse (100 /ig/ml) inhibits protein and RNA synthesis briefly
while delaying mitosis, division, and DNA synthesis. These single heat or CH pulses partially
synchronize mitosis, division, and DNA synthesis.
Experiments in which either 15-min HS or 10-min CH pulses were applied at different times
in selection-synchronized cultures have demonstrated several kinds and periods of sensitivity
to these agents. During roughly the first two-thirds of the cell cycle (measured between
divisions) mitosis, division, and DNA synthesis are delayed equally, delay increasing as a pulse
is applied progressively later in the cycle. The magnitude of the delay from a heat shock is
always greater than that from a CH pulse, but for both agents there is a period during which
delay is greater in magnitude than pulse length. The pattern of delay from cultures synchronized
by an induction method suggests that the period of increasing delay lies obligately within G2.
At 0-65 in the cycle the nature of the sensitivity to heat and CH changes. Between this transition point and the formation of a cell plate, CH has no effects on the timing of mitosis or cell
plate stage. However, CH can block the final splitting of the cell plate, leading to a permanent
cell plate and the formation of transient 4-celled pseudo-filaments upon resumed growth.
HS foOowing the transition point allows mitosis to be completed with normal timing to a stage
in which daughter nuclei occupy terminal positions in the cell, but formation of the cell plate
is delayed by about 30 min. Cells pulsed in the last third of the cycle may develop several
morphological aberrations. The cell plate is sometimes oblique or positioned at one end of the
cell, giving rise to daughter cells with 2 or no nuclei. Thus, it appears that some functions
related to normal positioning of the cell plate occur during the last third of the cycle.
Recovery from a heat shock applied prior to 065 in the cycle includes a period in which there
is increasing delay when a CH pulse is applied progressively later following the heat shock.
However, applied together, CH plus HS produce roughly the amount of delay due to the HS
alone. These facts are considered evidence for a common effect of both agents during the first
two-thirds of the cycle.
The results are discussed with reference to possible controls over several events of the cell
cycle.
• Present address: McArdle Laboratory for Cancer Research, University of Wisconsin,
Madison, Wisconsin, 53706, U.S.A._
2
M. M. Polanshek
INTRODUCTION
It is now known that many preparations for cell division occur and are completed
during cell cycles prior to a particular division in both prokaryotes and eukaryotes
(Mitchison, 1971; Slater & Schaecter, 1974). Among these preparations are general
requirements for some quantity of growth, expressed in terms of RNA and protein
synthesis, as well as a doubling of DNA during the 5-period in eukaryotes. More
specifically, the products of at least 32 genes are required for cell cycle traverse in
budding yeast (reviewed in Hartwell, 1974), and we suppose that these few dozen
products are components of one or more sequences of events which lead to division
(Mitchison, 1971; Hartwell, Culotti, Pringle & Reid, 1974).
A component of one sequence leading to division has been termed the 'heatsensitive' function (see Swann, 1957). This function was described in detail for the
ciliate, Tetrahymena, and relevant literature has been reviewed extensively (Zeuthen,
1964, 1974; Zeuthen & Rasmussen, 1971). The early observation was that a nonlethal shock of superoptimal temperature would delay or arrest division if it was
applied during roughly the first three-quarters of the cell cycle, with the inference
that a temperature-sensitive process required for division occurred throughout this
interval. When sensitivity ceased, the required preparation was believed either to be
complete or became heat-stable. The point at which insensitivity is achieved has been
called a 'critical point', 'point of no return', and 'transition point' (Mitchison, 1971)
The pattern of division delay prior to the transition point has received much
attention because it is the basis for theories of the control of cell division in which a
limiting synthesis or accumulation of substance needed for division spans a considerable portion of the cycle, and which has some properties of a clock (Rusch, Sachsenmaier, Behrens & Gruter, 1966; Sachsenmaier, Remy & Plattner-Schobel, 1972;
Bradbury, Inglis, Matthews & Langan, 1974; Zeuthen, 1961, 1974; Winfree, 1975).
In one paradigm, Tetrahymena, the key observation is that for a standard pulse
treatment, division delay becomes greater as a shock is applied later in the cycle.
Further, division delay is greater than the duration of a shock. One effect of the latter
is that division synchrony can be induced in an initially asynchronous culture because
cells must recover from a shock beginning a fixed time in the cell cycle before division.
This phenomenon has become known as 'excess delay' or 'setback'; it implies that
delay is greater than a given shock period without specifying a mechanism by which
this comes about. However, the standard interpretation of setback due to heat shock
is that heat discharges some cumulative preparations for division until cells reach the
transition point. Since the accumulation begins at a fixed point in cycle time before
division, a shock forces a population of cells to renegotiate division preparations together, which in turn constrains cells to proceed through division in synchrony
(Zeuthen, 1964).
The heat-sensitive preparation for division in Tetrahymena has not been unequivocally identified at the molecular level, but is generally believed to involve 'division
proteins' (Zeuthen, 1961). Evidence for this interpretation includes studies in which
division delay with a pattern similar to that for heat shocks has been effected with
Heat and cycloheximide effects onfissionyeast
3
inhibitors of protein synthesis or amino acid analogues, and for these agents a single
transition point has been found (Frankel, 1962, 1967a, b; Rasmussen & Zeuthen,
1962; Hamburger, 1962).
Our interest in the phenomena outlined above was to see how similar were effects
of heat shock and inhibition of protein synthesis on a fission yeast, Schizosaccharomyces
pombe. It has been shown that this yeast can be synchronized by heat shocks, and that
division delay increases during the cell cycle for heat-synchronized cultures (Kramhoft & Zeuthen, 1971). Also, in one strain of yeast, cycloheximide has been found to
delay division (Herring, 1973). A difference between this and previous work is that
we studied the effects of shocks on cultures of yeast synchronized by a selection
method, and we have completed more detailed analyses of effects on nuclear division,
cell plate formation, and various aspects of normal growth. Included is evidence that
both cycloheximide and heat affect more than one phase of the cell cycle, and evidence
consistent with the interpretation that both agents affect some processes in common.
MATERIALS AND METHODS
The fission yeast, Schizosaccharomyces pombe Lindner, strain 927b", was obtained from Professor U. Leupold, University of Bern, and was grown in Edinburgh Minimal Medium 2
(EMM2, Mitchison, 1970). The doubling time for cell numbers in exponential growth
(0-5-8 x io 8 cells/ml) at the normal culture temperature of 32 °C ia 140 min.
Synchronous cultures were usually prepared by the selection method of Mitchison & Vincent
(1965). Typically, cultures were harvested at 1-4 x i o ' cells/ml, yielding synchronous cultures
of O'S-2'5 x 10' cells/ml at time zero. The selection procedure is described in detail in Mitchison
(1970).
The induction method of Mitchison & Creanor (1971), using 3'-deoxyadenosine (Adr,
Sigma) to block DNA synthesis, was used for preparing synchronous cultures in several
experiments. The induction conditions for this strain of yeast are a period of 4 h in 3 mM Adr
in EMM2.
Division synchrony was usually monitored by estimating the cell plate index, which behaves
like a mitotic index (Mitchison, 1970).
Cell numbers were determined with a Coulter Counter Model B following light sonication
to break up cell pairs and clumps of cells (see Mitchison, 1970, for details).
Nuclear division figures can be seen in cells stained with Giemsa following acid hydrolysis
to remove RNA (Mitchison, 1970). The percentage of binucleate cells is based upon scoring
400 cells/sample. Our convention is to score as 'binucleate' all cells which show any stage
between the initially swollen, oval nucleus and that in which cells contain 2 nuclei but in which
a cell plate is not yet visible (stages 1-4 in Fig. 8).
Culture increase in dry mass was followed by measuring the absorbance at 595 nm (Am,6)
(Mitchison, 1970).
DNA was estimated colorimetrically using the diphenylamine reaction following Schneider
extraction, with 2'-deoxyadenosine as standard (Bostock, 1970). RNA was estimated from the
absorbance at 260 nm of appropriate dilutions of the hot perchloric acid extract from the
Schneider extraction (Munro & Fleck, 1966).
Relative rates of protein synthesis were estimated by incorporation of L-[4,5-'H]leucine
(Amersham) into acid-precipitable material during 10-min pulses of label. Typically, 1 ml of
culture was added to 1 /iCi [°H]leucine, 30 fig carrier L-leucine dissolved in 50 fi\ EMM2.
Incorporation was stopped after 10 min by addition of 1 ml of ice-cold 1 0 % (w/v) trichloroacetic acid (TCA). Samples were collected on Whatman GF/A filters, and assayed for radioactivity with a liquid scintillation counter.
Cell lengths were measured with a calibrated graticule and x 100 objective on cells dried on
to a slide, negatively stained with 10 % (v/v) India ink, and positively stained with 0-25 % (w/v)
crystal violet (Mitchison, 1970).
4
M. M. Polanshek
Heat shocks were applied by shifting a flask of cells from a water bath at 32 °C to another
at 41 °C. The pulse durations shown on graphs refer to the times at which transfers between
baths were made. The shock temperature is taken from experiments by Harnden (1957) and
Kramhoft & Zeuthen (1971). For most experiments, cultures of 10—25 Iri^ were shocked in
50-100 ml conical flasks to ensure a large surface area for heating and cooling, and the changes
in temperature took approximately 1 min. In larger cultures required for DNA determinations
(500 ml culture in a 3-I. flask), heating and cooling took as much as 5 min, and in some cases
the temperature rose to only 405 °C. However, the effect of shocks on cell number increase is
similar for both large and small culture volumes, so the speed of heating and the maximum
temperature reached are probably not critical within a few minutes or a degree.
Cycloheximide (CH, Sigma) solutions in EMM2 were always made up on the day of use.
The effective concentration of 100 /ig/ml was chosen after experiments showed that this was
the minimum concentration which produced maximum delay for io-min pulses of the drug
applied at about mid-cycle in cultures synchronized by selection (Polanshek, 1973). Pulses of
inhibitor were given by adding the drug solution to cultures in the volume ratio, 5 ml drug: 95 ml
culture. CH was washed out of cultures by collecting cells at room temperature (20 °C) on
0-45 grade Oxoid membrane filters with suction, rinsing with EMM2 at 32 °C, and resuspending
cells in fresh 32 °C EMM2. Controls, in which a pulse of medium was applied with identical
washing, showed that none of the parameters measured in this study were affected appreciably
by the pulsing procedure alone.
RESULTS
Effects of heat shock and cycloheximide on protein synthesis
In this study, effects of heat shock and an inhibitor of eukaryotic protein synthesis
(Siegel & Sisler, 1965; Cooper, Banthorpe & Wilkie, 1967; Rao & Grollman, 1967;
Baliga & Munro, 1971) have been compared. An assumption is that inhibitions
resulting from thermal stress can be understood by comparison with the effects of an
inhibitor whose mechanism of action is reasonably clear. Thus, we need to know
whether CH does in fact inhibit protein synthesis in this yeast, and how such inhibition
compares with that produced by elevated temperature.
Fig. 1 shows effects of 41 °C heating and 100/ig/mI CH on incorporation of pH]leucine into TCA-precipitable material during io-min pulses of label. CH cuts incorporation to 5-10% of the control rate of incorporation within 2 min, while heating
to 41 °C takes some 20 min to inhibit incorporation maximally. The maximum inhibition of pHJleucine incorporation by HS is slightly less than that achieved with CH.
The inhibition is reversible in either case when released by lowering the temperature
or washing out the drug. The lag which precedes recovery to the control rate of
incorporation increases with increasing pulse duration (Polanshek, 1973). For this
latter reason, and in order to fit many pulses into the cell cycle, short pulses of
10-20 min for CH or 15 min for HS were used. Experiments in which pulses were
applied at different times in selection-synchronized cultures showed that the kinetics
and degree of inhibition of pH]leucine incorporation and the kinetics of recovery
from either treatment match the data in Fig. 1, and do not vary during the cell cycle.
Effects of heat and cycloheximide on asynchronous growth
By analogy with systems such as that of Tetrahymena, one expects that any disturbance which releases a setback reaction will cause partial synchronization of division
in an initially asynchronous culture of cells (see Introduction). Figs. 2 and 3 show the
Heat and cycloheximide effects onfissionyeast
effects of single heat or cycloheximide pulses respectively on asynchronous cultures of
S. pombe. Prior to either pulse, growth is characteristic of asynchronous cultures of
yeast. Following either pulse, cell number increase is depressed up to the partially
synchronous division, which is reflected in the cell plate index, percentage binucleate
A
^— a
1500
-
-
1000
1
c J2
O
w
500
-
1
1
1
1
1
ZJ
CL
0
8
b
D
1500
_
B
3 -O
QJ _
^ %
it
1000
500
-
A
V
n
/
%
y
o
e
30
u
O
60
90
Time, mm
Fig. 1. Effect of 100 fig/ml cycloheximide (A) or 41 CC heat shock (B) on L-fH]leucine
incorporation into acid-precipitable material during 10-min pulses of label. Details of
the pulsing procedure are in Materials and methods. Pulses began at the times indicated by the symbols. Controls, • ; continuous treatment with either CH (A) or heat
(B) beginning at time = o, OI recovery from a 10-min CH pulse (A) or a 15-min heat
shock (B), • .
cells, and increases in DNA and cell numbers. Growth then returns to normal. For
the CH-treated culture, the burst in division comes about 70 min after the end of the
pulse, while a similar burst follows the heat shock by roughly 90 min.
Increases in the A69S and RNA/ml are hardly affected by CH, but A^g increase is
markedly inhibited following a heat shock. This observation indicates a decrease in
the accumulation of dry mass by heat-shocked cells. Consistent with this interpretation
5
6
M. M. Polanshek
is the fact that cells do not appear to grow in length for 30-45 min following a heat
shock when viewed in time-lapse films (Polanshek, unpublished). Since the cell wall
material must be a large fraction of the total cell mass, it is probably inhibition of this
aspect of growth which accounts for the depression in the A ^ following heat shock.
Fig. 2. Effect of a single 15-min, 41 °C heat shock (bar, 1—1) on growth of an asynchronous culture of yeast. The curve for the percentage binucleate cells (dashed
line) is based upon other experiments because mitotic cells were not scored in this
case. DNA, A ; RNA, • ; cell number, A J Absorbance695 Tim, • ; cell plate index, O-
A slower increase in dry mass is also reflected in measurements of cell length at the
times of the cell plate peaks in Figs. 2 and 3. Sincefissionyeast are roughly cylindrical,
a measurement of length is proportional to cell volume, so the histograms presented
in Fig. 4 can be read for the trend in either property. Cells at division are roughly
the same length on average for heat-shocked or control cultures, but the distribution
of lengths is somewhat broader in the treated culture, with some rather long dividing
cells. Indeed, the size distribution of dividing cells in the shocked culture is rather
like the distribution of sizes for random cells in the control population. This suggests
that all cells of the population contributed proportionately to the decrease in dry
mass seen in Fig. 2.
There are some similarities in the effects of HS and CH on cell length at division.
Fig. 4E-F shows the length distributions for control and CH-treated cells at the cell
plate peak in Fig. 3. The length distributions are again somewhat broader in the
treated culture than in the control. In contrast with the HS effects, the mean cell
lengths are markedly greater in the CH-treated culture than in the control. This fact,
Heat and cycloheximide effects onfissionyeast
together with the minor depression in the \ K following the CH pulse (Fig. 3),
implies that culture growth measured so broadly has been little affected by the CH
pulse, and cells have managed somewhat more growth in size and mass than is usual
at the time of division.
Summing up, the data in Fig. 4 suggest that cell lengths are not standardized when
cell division is partially synchronized as a result of HS or a CH pulse. Thus, alignment
of division bears no direct relation to cell volume (see Faed, 1959; Mitchison &
Creanor, 1971).
Fig. 3. Effect of a single 10-min, 100 jUg/ml cycloheximide pulse on growth of an asynchronous culture of yeast. The position of the pulse is indicated by the bar (h-1). RNA,
• ; DNA, A ; cell number, A ; AbsorbanceM6 „„, • ; cell plate index, O ; binucleate
cells, • .
Division delay in synchronous cultures
Since cell division is partially synchronized as a result of a single pulse of either
heat or cycloheximide, we expect that division delay due to a standard shock will
increase as the shock is applied progressively later in the cycle (Mitchison, 1971;
Kxamhoft & Zeuthen, 1971). Fig. 5 shows the results of experiments in which either
a heat shock or CH pulse was applied at several times during the cell cycle in cultures
synchronized by selection. Pulses given before about 0-40 in the cycle result in single
cell plate peaks occurring somewhat later than the controls. Later pulses split synchronous cultures into 2 groups of cells which can be described from their respective
cell plate peaks. The earlier peak is interpreted to include cells which have escaped the
delaying effects of a pulse because they are past a transition point for division delay
which is late in G2. The later peak represents cells which were sensitive to a treatment
7
8
M. M. Polanshek
because they were positioned in the cycle prior to the transition point at the time of the
pulse. The escape peak increases as the peak of the delayed population declines, there
being first few and finally all cells in the escape peak. The transition point for division
delay occurs at about 0-65 in the cycle from the fact that a pulse applied at this time
splits a synchronous culture into 2 numerically equal groups of cells.
50 -,
11 88±100
50 -,
905±1 90
2 50!
11-10±1-07
F
8-40±2-16
12-70±1-43
11 75±1-63
v
9S3±1 60
6
12
18
6
12
18
Cell length, pm
Fig. 4. A—H. Length distributions and mean lengths for cells at the times of the cell plate
peaks in Figs. 2 (A-D) and 3 (E-H). 100 cells were sized for each histogram from random
fields of cells stained with crystal violet on an India ink background (see Materials
and methods), A, E, controls, cells with cell plates only; B, F, controls, random cells;
C, G, cells with cell plates only, after HS (c) or a CH pulse (G) ; D, H, random cells, after
HS (D) or a CH pulse (H). Figures given are mean length ± standard deviation.
From a series of pulsing experiments a curve of division delay as a function of the
position of a pulse in the cell cycle can be drawn. Delay is estimated as described in
the Appendix. Fig. 6A,B gives delay curves for heat shocks and CH pulses, respectively.
For both agents there is a period of increasing delay over a large fraction of the cycle.
In the case of CH pulses, delay is zero or a few minutes for escape peak cells, but cells
past the transition point for increasing delay are still delayed roughly 30 min by a
heat shock. While delay is greater in magnitude and begins earlier for heat shock, the
transition point for the phase of increasing delay is approximately 0-65 in the cycle
for either agent. For at least part of the period of increasing delay, both heat and CH
produce excess delay (see Introduction).
When cell plate formation is delayed during the major period of increasing delay,
division as measured by increase in cell numbers is also delayed, as is DNA synthesis
Heat and cycloheocimide effects on fission yeast
(Polanshek, 1973, and see Figs. 2 and 3). However, the earliest event which we can
show to be delayed is nuclear division. This process is detectable in Giemsa-stained
preparations by about 0-75 in the cycle, as estimated from the peak of dividing nuclear
figures which occurs roughly 15 min before the peak in the cell plate index. Fig. 7
shows control and shocked synchronous cultures in which mitosis was followed. For
the CH-treated culture (D), nuclear division is delayed along with cell plate formation,
Cycloheximide
Heat shock
Control
Control
30 r
20
30
20
10
10
0
40
0
:
°H
30
29
30
i\
20
20
10
2 10
f- 0
0 50
H
8 30
0
qft
Tl
20
^ 20
10
n
40
10
1
1
1
,^-rPoO
1 °
0
30
20
20
10
10
0
0
Time, h
Fig. 5. Effect of a 15-min, 41 °C heat shock or a 10-min, ioo/ig/ml CH pulse on the cell
plate index in selection-synchronized cultures of yeast. Shocks, indicated by bars ( M ) ,
were applied at 3 different times between the first and second divisions following
selection. The position of each shock is given as a fraction of a cell cycle between
divisions, assuming that the times of half-doubling in cell numbers occur 15 min
(o-io of the cycle) after the mean times of the cell plate peaks for control cultures.
Mean times for cell plate peaks are indicated by arrows, and were determined as
described in the Appendix.
but remains in the same relative position prior to the cell plate peak as in the control (c).
When there are delayed and escape peaks of cell plates, as shown, the population is
split with respect to the time of nuclear division as well, suggesting that after the
transition point for increasing delay, nuclear division proceeds with normal tuning,
and cells enter into cell plate stage as in untreated cultures. This is confirmed for
cultures left in CH, as cells continue to enter cell plate stage for roughly 45 min
9
M. M. Polanshek
IO
when cycloheximide is present, and mitotic figures decrease to zero (Polanshek, unpublished).
The picture for heat-shocked cultures is more complex. First, nuclear division is
delayed together with cell plate formation during the period of increasing delay. As
with CH, there is a single peak in both the percentage binucleate cells and the cell plate
.
.
,
.
I
.*•
•
.
.
i
i
.
,
T
,
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.
05
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>:
—
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Mid -point of heat shock
ui
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i
.
.
,
.
.
I
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|
I
i
i
o
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30 &
20 -
—I 20
"
—| 10
10
0
O0
P'
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i i 1 A
0-5
^J^*
. . ! . . . .
10
°° ° n ^
-10
1°.*. . ,
0-5
10
(0 0)
Mid-point of CH pulse
Fig. 6 A, B. Division delay in yeast cultures synchronized by selection as a function of
the position of a is-min, 41 °C heat shock (A) or 10-min, 100 fig/ml cycloheximide
pulse (B) in the cell cycle. Shocks were applied between the time of selection and the
second cell plate peak. 00 and 1 -o in the cycle are the times of half-doubling in cell
numbers, so the data are plotted for a cell cycle running between divisions, not between
cell plate peaks. Filled circles represent cells delayed in the major period of sensitivity to heat or CH (see text), while open circles represent 'escape peak' cells
which are past a transition point for division delay at 0-65 in the cycle (arrows). The
curves are drawn by eye. 'Excess delay' is total delay minus the length of a pulse
treatment or shock (see Introduction).
index early in the cycle, while later pulses split the population. Fig. 7 shows data for
such a population split into escape and delayed fractions, and it is clear that there are
also 2 peaks of binucleate cells. We have noted that cell plate formation is delayed
roughly 30 min following the transition point for increasing delay. However, nuclear
division occurs at the normal time compared with the control for those cells which
Heat and cycloheximide effects on fission yeast
II
form the escape peak of the cell plate index curve. This means that nuclear division
and cell plate formation have been separated by about 30 min longer than in the normal
temporal sequence.
Transition points and periods of delay
We can reconstruct the events which occur following the first transition point in
somewhat greater detail from slides stained for mitotic nuclei. Such a reconstruction is
given in Fig. 8. Representative morphologies of cells at various times in the cell cycle
were traced from photographs, and represent the normal course of growth, mitosis,
30
20
20
10
10
20
0
20
10
10
LIT*.
Time, h
Fig. 7. Effect of heat shock or cycloheximide on mitosis. Cell plate index, O ; binucleate
cells, 0 . A, control for heat-shocked culture (B); C, control for CH-pulsed culture
(D). Positions of pulses are shown by bars (h-i). Arrows indicate mean times for the
several peaks. For both controls (A, C), the mean time of the % binucleate peak is about
15 min before that for the cell plate index. In (D), a CH pulse has split both the % binucleate and cell plate index curves into 2 peaks. However, the % binucleate peak
remains 10-15 min prior to the cell plate index peak for each of the 2 sub-populations.
In (B), the heat shock has split the culture into 2 major cell plate peaks and 2 %
binucleate peaks. The second % binucleate peak represents cells which form cell plates
at the second major cell plate peak (3), and these experience severe division delay;
nevertheless, the % binucleate peak is about 15 min prior to the cell plate peak. The
first peak of binucleate cells represents cells which form cell plates at the first major
cell plate peak (2), but the time between these peaks is roughly 40 min, or 25-30 min
more than in controls. Thus, mitosis has been separated from cell plate stage by 2530 min extra for these escape peak cells. The cell plate peak at (J) in (B) represents a
small fraction of the culture which was at or near the end of mitosis at the time of the
heat shock, and which formed cell plates during and after the shock.
M. M. Polanshek
12
and division (see also McCully & Robinow, 1971; Johnson, Yoo & Calleja, 1974).
The total cell cycle is taken to be the 140 min characteristic of growth in EMM2 at
32 °C, and o-o and i-o in the cycle are the times of division denned operationally from
cell number increase measured with a Coulter Counter after sonication (Mitchison,
1970).
1 2
3 4
05
Aberrant cells
I M I G,
A
A
Heat
shock,
15 mm,
41 °C
Delay of mitosis, cell plate
formation, division, DNA
synthesis.
Delay Increases as pulse Is
jpplcd progressively later
Cycles
Delay as above for heat
hexlmlde, shock, but of lesser
10 mm,
magnitude
100 //g/ml
Mltmli
torn pitted
i tuxe 4
Ith normal
tlmln|. call
pttte
(ornutlon
deliyed
30 min
s I
DlYlikm
r
tnbkckod
c*l|, with
» cell
NodlvlUon delay,
except thit K>m» cdli
ccJIoct In cell pUta
i u | t . which may t *
lrre*«nibiy blocked
Fig. 8. Summary of heat and cycloheximide effects during the cell cycle. The cycle of
approximately 140 min is plotted in fractions of a cycle between divisions, and is also
broken into the conventional periods of S, G%, M, Gx (Howard & Pelc, 1953). Outlines
of cells with nuclei were traced from photographs, and those above the cell cycle map
represent the normal course of growth, mitosis, and division. Effects of heat and
cycloheximide pulses are summarized for periods of sensitivity indicated by the width
of the boxes and the arrowheads. Cells with aberrant morphologies were also traced
from photographs.
The evidence for a major period of increasing delay due to pulse treatments has
been presented earlier. Cultures are observed to be split into either of 2 populations
when pulses are applied late in the cycle, and this is the main evidence that a transition
point exists at 0-65 and behaves as a point. That is, cells very suddenly become insensitive to increasing division delay, and do not gradually lose their sensitivity over
a measureable fraction of the cycle. However, for CH there must be a second transition point during cell plate stage. The evidence for this is that the cell plate index roughly
doubles during the first 30-45 min of continuous exposure to CH in asynchronous cultures, or for approximately the amount of time between the first transition point and cell
plate formation (Polanshek, 1973). Further, a normal preparation for cell plate completion must fail during this part of the cycle because most of the cells which accumulate
with cell plates in the presence of the drug do not separate at these cell plates, but remain attached during subsequent growth when the drug is removed. If a culture remains
in CH, the cell plates which form thicken and become visibly double and roughly
elliptical, with the 2 sides of the cell plate connected at the circumference of the cell.
Heat and cycloheximide effects onfissionyeast
13
The impression is that a cell plate may be completed in the presence of CH, but
splitting at the margins cannot be accomplished. The result of resumed growth of
cells with double cell plates is the production of 4-celled filaments in the configuration
drawn in Fig. 8. These filaments are transient since daughter cells at either end split
off normally, though not usually in synchrony.
Heat shocks produce more complex effects during the cycle, and induce more varied
aberrant cell types than do CH pulses. Shocks applied before the transition point at
0-65 delay the events of division which follow the transition point, but mitosis, cell
plate formation, DNA synthesis, and division are then completed in normal sequence
and timing. Following this first transition point, mitosis, once begun, seems to occur
normally at the shock temperature until stage 4, Fig. 8. During a shock, the percentage of cells in early stages of nuclear division declines as there is a reciprocal increase in the percentage of cells in stage 4 (Polanshek, 1973). Once collected at this
stage, the nuclei remain at the extreme ends of the cell even though a cell plate forms
roughly 30 min after the heat shock has ended. Normally, the cell plate would appear
immediately after, or even during, stage 4. Thus, there is apparently a block late in
nuclear division, and this can be considered a second transition point for thermal
shock.
Between the first and second heat transition points, shocks delay cell plate formation
roughly 30 min. Shocks during this period also result in a number of aberrant morphologies, shown in Fig. 8. These configurations may be interpreted as mistakes in the
placement of the cell plate such that nuclei either are not sorted into different cells,
or are sorted into cells of markedly unequal size. It is not known whether small segments containing single nuclei are viable cells, but some of the small segments appear
highly refractile, and are probably dead.
Effect of cycloheximide on recovery from heat shock
In view of the similarities in the pattern and period of sensitivity of cells to division
delay during the first two-thirds of the cell cycle, we may suppose that both heat and
CH affect common processes leading toward division; evidence that division delay
from the 2 treatments applied together is not equal to the sum of the delays for either
treatment alone would be consistent with this idea. The reasoning is that either
treatment should discharge the same preparation for division so that the total delay
from a combined treatment should be equal to that for the single treatment which
alone produces the longer delay. One experiment in which this argument was tested
is shown in Fig. 9. Here the shocks were sequential, with the heat shock first. Other
experiments gave similar results when both treatments were applied coincidentally.
It is clear that delay is not additive in this example, since an additional CH pulse
added only a few minutes to the delay induced by the heat shock, though CH alone
produced 22 min of delay.
Because of this last result, experiments were done to see whether there was a phase
of increasing delay when CH pulses were applied at different times following a single
heat shock. The assumption was that if heat shock effects included CH-blocked events,
then a potential for increasing delay from CH pulses would be part of the process of
2
CEL 23
M. M. Polanshek
20
Control
10
0
40
39 mm
30
20
u,
10
ID
n
jj- 0
g 30
46 min
20
10
0
22 mm
20
10
1
2
Time, h
3
Fig. g. Effect of combined 15-min, 41 °C heat shock and 10-min, ioo/*g/ml cycloheximide pulse on division. Pulses were applied as shown by bars (l—l), and positions
of cell plate peak mean times are indicated by arrows. Pulses were applied between
selection of cells and the first synchronous division. The division delays given on figures
were estimated as described in the Appendix.
00
05
10
Mid-point of CH pulse
Fig. 10. Division delay for cycloheximide pulses applied at different times following
a heat shock in cultures synchronized by selection. The position in the cycle of the 15min, 41 °C heat shock is indicated by the bar (1—l); 10-min, 100 /tg/ml CH pulses
applied at times shown by points resulted in delayed (#) and 'escape peak' populations of cells (O)- All pulses were applied prior to the first division following selection.
Delay due to the heat shock has been subtracted from the total delay to determine net
CH delay. Arrow indicates time of CH transition point.
Heat and cycloheximide effects onfissionyeast
15
recovery from heat shock. Three such experiments are summarized in the division
delay curve in Fig. 10. The predicted phase of increasing delay is apparent, but the
magnitude of the delay is less than that found for CH pulses alone (Fig. 6B). This
may reflect the fact that cultures are delayed progressively less by shocks subsequent
20
10
0
20
"a.
s
°
0
30
20
10
-Adr
1
2
3
Time, h
Fig. n.Effectof a is-min,4i CC heat shock on the cell plate index following synchronization of a culture with 3'-deoxyadenosine (Adr). Heat shocks were applied at the times
indicated by bars (l—i). Approximate mean times for cell plate peaks are shown by
arrows.
to the first one for either CH or heat, eventually dividing despite a series of several
shocks (Polanshek, 1973). There is evidence in Fig. 10 for a transition point for CH
delay, since populations are again found to be split into escape peaks and delayed
peaks by the CH pulse. The CH transition point, based upon these few experiments,
has been delayed by roughly the 20 min maximum division delay produced by CH.
i6
M. M. Polanshek
Division delay in cultures synchronized by induction
All of the experiments described so far were performed with either log-phase,
asynchronous cultures of yeast, or with cultures synchronized by selection of small,
young cells. One of the more powerful methods for studying cell cycle events is to
disturb the cycle to see which events and temporal spacings can be altered, and which
remain obligatory. Thus, the effects of shocks on cultures synchronized by a second
method are of interest.
i
i
i
i
i
i
i
i
I
-
40
20
c
i
>;
/
30
•S
3
30
/
20
/
10
• /° °
/*
/
°
/ *
10
c
1
|
-o
/
-
-10
r>
-Adr
30
60
90
120
150
Mid-point of heat shock, mm
Fig. 12. Summary of division delay for is-min,4i "C heat shocks applied to cultures of
S.pombe synchronized with a 3'-deoxyadenosine block (see Fig. 14). Delay was estimated for the first cell plate peak after removal of the block. Solid circles are delayed
peaks, open circles indicate 'escape peaks' (see Fig. 6 for similar data on cultures
synchronized by selection).
The synchrony procedure chosen for comparison is that of Mitchison & Creanor
(1971) in which an asynchronous culture is treated with 3'-deoxyadenosine (Adr) to
block DNA synthesis. This leads to division synchrony upon release from the block,
and a long Gx period is created while G2 becomes short compared with the G2 seen
in a culture synchronized by selection. A characteristic of this synchronization procedure is the production of oversized cells which go through 2 abnormally closely
spaced divisions some time after release from Adr. It has been argued that the reason
that the 2 divisions are so close together in time is that division proteins accumulate
during Adr treatment, allowing 2 rapid division cycles once DNA synthesis has caught
up with the level of division proteins (Mitchison & Creanor, 1971). We have assumed
that division delay resulting from heat shock assays for the same preparations for
division under conditions of Adr synchrony as were measured in cultures synchronized by selection. Division delay has therefore been measured for pulses applied
between the end of the Adr treatment and the first synchronous division.
An experiment of the latter sort is shown in Fig. 11, and a series of such experiments is summarized in Fig. 12. Two facts are clear. First, division delay has a different
pattern here than for cultures synchronized by selection in that there is a period in the
cycle when a heat shock produces no division delay. Delay of the first division begins
to increase shortly after DNA synthesis has resumed (see fig. 2 in Mitchison &
Heat and cycloheximide effects onfissionyeast
17
Creanor, 1971). This suggests that heat-sensitive division preparations do not
start until at least the beginning of 5-phase. If the latter is correct, sensitivity
to increasing division delay belongs in the G2 phase of the the cell cycle, with possible
overlap back into S-phase; this is close to what is found for cells synchronized by
selection.
A second point is that the time between the 2 synchronous divisions is not increased
when the first division is delayed. This suggests that heat-sensitive preparations for
the second division are not executed prior to the first division. An implication is that
division proteins have not been accumulated and sequestered during the time that
cells were in Adr, as has been suggested (Mitchison & Creanor, 1971).
DISCUSSION
Various factors and processes have been identified as determinants of cell division
in eukaryotes (see Mitchison, 1971, and Introduction). In this paper we have concentrated on processes which limit division when they are disrupted by heat shock or
the protein synthesis inhibitor, cycloheximide; as a result of the disruption, cells
experience division delay. The paradigm is a system in which cultures of the ciliate,
Tetrahymena, can be brought into division synchrony by a series of heat shocks
(Zeuthen, 1974). Such synchronization has been explained using division delay data,
and theories of the control of cell division derived from these data relate broadly to
ideas about possible initiators of mitosis (Mitchison, 1971). Our interest was to see
how closely parallel are the responses of a fission yeast, Schizosaccharomyces pombe,
and the ciliate to heat shock and inhibition of protein synthesis.
In the study most comparable to this one, Kramhoft & Zeuthen (1971) found a
period of increasing delay for heat shocks in the cell cycle of yeast previously synchronized by heat shocks. The important points for comparison are the facts that in cultures
synchronized either by selection (this study) or heat shock, increasing division delay
and a transition point ending this period of sensitivity can be demonstrated. Additionally, delay due to a pulse is greater than pulse length during much of the cycle. The
period over which there is increasing delay is comparable in length, and the maximum
delay of 30-40 min for 15-min heat shocks is roughly the same as we find for the first
of 3 periods of sensitivity described here (Fig. 8).
Having confirmed that heat shocks produce a pattern of increasing excess delay in
yeast, the question was whether pulses of CH produce a similar pattern, as would be
expected from a 'division protein' explanation of division delay (see Introduction).
The period of increasing delay which we have demonstrated for CH pulses during
the first two-thirds of the cycle fits this expectation. Also, delay from heat shock and
from CH is not additive, and recovery from heat shock includes a potential for increasing division delay which is revealed when CH pulses are applied following a heat
shock. These facts are consistent with the interpretation that both agents discharge
a common preparation for division, as in the Tetrahymena division protein scheme
(Zeuthen, 1974). However, heat shock and CH inhibit both protein synthesis and other
macromolecular syntheses. Additionally, the difference in the magnitude of division
18
M.M. Polanshek
delay produced by the 2 agents suggests that heat shock is either more potent than
CH while affecting comparable processes, or that, among its effects on cells, heat shock
affects processes which are also CH-sensitive. A similar problem in interpretation
occurs with ^-fluorophenylalanine-induced delay in Tetrahymena, where this drug
produces greater delay than heat shock early in the cycle (Zeuthen, 1964; Mitchison,
1971). The fact that heat severely inhibits growth of fission yeast in length and in dry
mass suggests that recovery from heat shock includes more than simply resupply of
division proteins. To a first approximation, heat shock delay during the first two-thirds
of the cycle can be accounted for as the sum of a variable increasing delay equal to
that produced by CH alone and a constant delay roughly equal to the duration of a
heat shock. The constant delay is perhaps reflected in the reduced rate of growth in
cell length following heat shock. Thus, heat delay may be due to an effect on particular
syntheses required for cell cycle traverse (division proteins?) and to a more general
suppression of cellular metabolism.
Although it has been argued that division proteins are required for structures such
as the mitotic apparatus (Zeuthen & Williams, 1969; Mitchison, 1971), other data
suggest that a pattern of increasing delay with a transition point is not characteristic
of one homologous sensitive process in various systems, but may rather relate to
common and evolutionarily old patterns of cellular metabolism. For example, in
Chinese hamster cells treated during Gx with pulses of either cycloheximide or puromycin, initiation of DNA synthesis is delayed with a pattern of increasing excess
delay up to a transition point 55 min prior to 5-phase (Schneiderman, Dewey &
Highfield, 1971; Highfield & Dewey, 1972). Further, it is hard to find an analogy with
eukaryotic structural elements in Escherichia coli, where increasing division delay
occurs due to heat shocks and p-fluorophenylalanine pulses (Smith & Pardee, 1970).
It may be that the accumulation of substances (initiators?) to threshold levels is the
common mechanism which may or may not be sensitive to heat shock and to interruption of protein synthesis; there is evidence for initiators of DNA synthesis in
bacteria and mammalian cells (Donachie, Jones & Teather, 1973; Highfield & Dewey,
1972), and of mitotic initiators in the eukaryotic slime mould, Physarum (Sachsenmaier et al. 1972; Bradbury et al. 1974). Alternatively, interruption of the normal cell
cycle may require that a whole programme of biochemical synthesis be repeated, as in
the ' quantal control' idea developed to explain the recapitulation of enzyme synthesis
which occurs following disruption of an aggregation stage in Dictyostelium (Newell,
Franke & Sussman, 1972). There is some evidence that cells repeat sequences of events
from the cell cycle in which they were delayed: Tetrahymena reconstructs an oral
apparatus following heat shock, and fission yeast repeats a period of susceptibility to
cycloheximide division delay.
A second period of heat-sensitivity for fission yeast covers the period of mitosis.
During this period, cycloheximide has no division-delaying effects, and we conclude
that abrupt and nearly complete inhibition of protein synthesis does not interfere
with mitosis once it has begun. While mitosis proceeds at the higher temperature,
formation of the cell plate is delayed. From the work of Johnston et al. (1974), we can
propose that heat interferes with the formation near the end of mitosis of the annular
Heat and cycloheximide effects onfissionyeast
19
rudiment, the template from which centripetal growth of the septum occurs. If
formation of the annular rudiment were disrupted by heat shock, nuclear division might
be completed on time though cell plate formation could be delayed. This would not
account for the fact that nuclei remain at the extreme ends of the mother cell when a
shock occurs during mitosis. It is, however, well established that spindles and similar
structures are disrupted by high temperatures (Marsland, 1970). If daughter nuclei are
pulled toward the middle of the presumptive daughter cells by the spindle, they might
not suffer their normal final positioning at high temperatures.
We have presented some evidence that the position of the cell plate can be affected
during this second phase of heat sensitivity, before there is any trace of a septum. It
is possible that sites at which a septum may form are integrated into the membrane or
wall at regular intervals, as in bacteria and budding yeast (Donachie et al. 1973; Cabib
& Farkas, 1971). If heat activates such sites, perhaps at random and possibly prematurely, non-central cell plates could be formed. The usually strict relationship of a
central plane of fission perpendicular to the mitotic apparatus breaks down in these
circumstances.
The last section of the cell cycle in which there is a distinct effect of inhibitors on
a particular event is the cell plate stage. Heat shock can fix a cell in this stage for an
indefinite period; cycloheximide has clear, sequential effects on cell plate formation,
In the presence of CH, cell plates form as usual, but the septum fails to split at its
circumference. The central scar plug layers of the cell plate apparently split apart,
leading to an elliptical double cell plate with daughter cells attached at the annular
rudiment. It is likely that autolysin-like enzymes are required to break the old wall at
the annular rudiment after the cell plate is completed (Johnson et al. 1974). A tentative conclusion is that CH interferes with such autolytic functions, possibly by blocking
the synthesis of the enzymes. Once autolysis has been blocked, the fault is not rectified
in most of the cells which have suffered the blockage. This suggests that autolytic
enzymes may be available only briefly once each cycle, and that they must be available
at a specific stage in cell plate maturation. It is also possible that the peripheral wall
is not a suitable substrate for lytic enzymes once the cell plate has thickened excessively.
The several periods of sensitivity which we have described in the cell cycle of fission
yeast have all been related to interference with processes obviously associated with
cell division. These periods are separated from one another by transition points.
A transition point traditionally has been defined as a single time in the cell cycle at
which sensitivity, usually measured as blockage of division or mitosis, ceases for a
particular agent or class of agents, such as inhibitors of protein synthesis (Mitchison,
1971). However, multiple transition points for division can be found in many systems
when such points are defined as times at which division sensitivity changes abruptly.
For example, there are 2 periods of susceptibUity to ultraviolet-induced division delay
in fission yeast, with sharp transition points between them (Gill, 1965). In Tetrahymena, there is a heat transition point at the end of the period of increasing division
delay, but a second one at a late fission stage, since fission is disrupted by heat shock
applied while it is occurring (Zeuthen, 1964).
Transition points may identify times of change in the physiology of cells, especially
20
M. M. Polanshek
when agents block some events very rapidly. Nevertheless, a transition point for energy
metabolism in sea-urchin eggs was shown by Epel (1963) to be artifactual when inhibition of respiration was incomplete, as in early studies (see Swann, 1957). It is
possible that such a problem might occur when using inhibitors of protein synthesis.
For example, Chinese hamster cells can move from G2 to mitosis when small amounts
of leucine are added to leucine-deficient medium, though these cells are blocked in
G1 with the same amount of leucine (Everhart & Prescott, 1972). In gut epithelium of
the rat, cells move from G2 into mitosis when there is an 80 % reduction in protein
synthesis (Verbin, Liang, Saez, Diluiso, Goldblatt & Farber, 1971). These examples
suggest that proteins required for cell cycle traverse are made even if the total capacity
for protein synthesis is well below normal. This must also be true for S. pombe,
because cells will go through one or two division cycles when deprived of a nitrogen
source (Faed, 1959, cited in Mazia, 1961), and cells make progress toward division
which can be negated by CH following a heat shock, when the rate of protein synthesis
is reduced.
I would like to thank Professor J. M. Mitchison and Drs R. G. Burns and J. Creanor for
their help and advice during the course of this study, and the many people - especially (in
addition to the above) Drs R. S. S. Fraser, M. E. Rogers, H. C. Bennet-Clark, R. A. Kille,
Miss P. Aitchison, Miss C.Wilson, Mr D. Cremer, and Mr N. McKay - who made the
Zoology Department at Edinburgh such a friendly place during my three years there.
It is a pleasure also to acknowledge the influence and inspired teaching of Professor Daniel
Mazia of the University of California, Berkeley, who directed me first to Professoi Mitchison's
course, thence to Edinburgh.
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[Received 25 May 1976)
SIEGEL,
APPENDIX: ESTIMATION OF DIVISION DELAY FOR S. POMBE
FROM CELL PLATE INDEX CURVES
In cultures of yeast synchronized by selection (Mitchison & Vincent, 1965), the cell
plate index curve is generally symmetrical (Fig. 13 A). The curve broadens and the
maximum becomes smaller at succeeding cell plate peaks as division synchrony wanes.
Assuming that all cells take, on average, the same amount of time to complete cell plate
stage, the mean time of mid-cell plate stage for the population can be estimated by
finding the time at which the area under the cell plate peak is halved; for a normal
curve this mean time will be the time of the maximum value of the cell plate index.
When an agent is applied which causes division delay, the cell plate index curve is
displaced to the right. For heat shock or cycloheximide, pulses early in the cycle displace the whole cell plate peak, and the mean time of mid-cell plate stage can be estimated as for a control culture (Fig. 13 A). Then the mean time for the control cell plate
peak is subtracted from that for the treated culture, giving the magnitude of division
delay.
Pulses applied relatively late in the cell cycle split the cell plate index curve into 2
(or more) peaks. With a split culture it is inaccurate to compare the time of the cell
plate peak for either sub-population with the mean time for the whole population. We
have used a graphical method for estimating division delay by comparing the time of
the cell plate mean for each cell plate peak in a treated culture with the mean for the
corresponding fraction of the control population. With this method one eliminates an
Heat and cycloheximide effects onfissionyeast
artifactual phase of increasing division delay for escape peak cells which is generated
when the times of the escape peaks are compared with the time of the control cell plate
peak. One also eliminates negative values of delay for escape peak cells.
Time, h
Fig. 13. A and B are cell plate index curves from control and cycloheximide-pulsed
cultures, respectively, and were taken from Fig. 5 in the main text. Arrows mark the
estimated mean times for mid-cell plate stage for the various cell plate peaks, and the
bar (l—l) shows the position of the cycloheximide pulse.
To estimate the mean time of mid-cell plate stage for each peak, we first smooth in
the curves, as in (c) and (D). The shapes of the curves for the pulsed culture will be
assymetric as drawn in (D) if the pulse split the culture at a transition point or very short
transition interval. The control population is assumed to be the union of the 2 populations found for the pulsed culture. If the CH pulse has not altered the duration of cell
plate stage for treated cells, then the areas under the two cell plate peak3 in D should
sum to give the area under the control curve in c. The 2 cell plate peaks in D are
fitted under the control curve in c by aligning the left hand edge of the left peak (fine
stippling) with the left hand edge of the control, and the right hand edge of the right
peak (large stipples) with the right hand edge of the control. Note that the sum of the
2 stippled areas is approximately equal to the area under the control. We can now
estimate when the cells in each peak of the pulsed culture would have divided in the
control. The mean time of mid-cell plate stage is estimated by finding the time which
splits the area under each peak in half; these times are found for both peaks in the
pulsed culture and for the same peaks fitted under the control in c. Subtracting the times
found for the peaks under the control curve from the times found for the peaks in D,
we estimate that the first peak in D is displaced 3-5 min from the position of the same
fraction of the control population. Similarly, the second peak in D is delayed 18-20 min
compared with the corresponding fraction of the control.
The precision of this method of estimating division delay is indicated by the spread
of division delay values in Fig. 6 of the main text.
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