235
FEMS M~robiologyLetters 2 (1977) 235-237
O CopyrightFederationof EuropeanMicrobiologicalSocieties
PubUsh~dby Elsevier/North-HollandBiomedicalPress
CELL SIZE AND C E L L DIVISION IN Y E A S T C U L T U R E D A T D I F F E R E N T G R O W T H R A T E S
M.N. JAGADISH+ A. LORINCZ and B.L.A. CARTER
Department of Genetics', Univeraty of Dublin, Trinity College, Dublin 2, Ireland
Received 28 July 1977
1. Introduction
For a wide variety of cell types cells at division display a rather constant cell size from generation to
generation. If cell division is temporarily arrested cell
size at the subsequent division is larger than normal
but within a few generations of the arrest the prearrest size at division is restored [1]. It is likely that
there is coordination between the processes of growth
and cell division which maintains this constancy.
We have observed that the mean size of Saccharorayces cerevisiae is related to growth rate: the faster
the growth rate the larger the cells. We have investigated the nature of this variation in the hope that it
would reveal something of the relationship between
growth and division. Our results suggest that growth
and division are coordinated by the requirement that
yeast cells attain a critical cell size before an event on
the pathway to cell division can be completed.
elements and vitamins as described previously [3].
The media for chemostat cultures contcdned, per litr~:
yeast extract, 10 g; bactopcptone (Difco), IO g; ~ucose (as growth-limiting nutrient), 10 g; ammonium
sulphate, 5 g; ammonium phosphate, 2 g; magne:fium
sulphate, 1 g; adenine, 0.1 g;pH 5.5. The temperature
was maintained at 24°C.
2.2. Measurement o[ cell growth and cell size
Increase in cell number was measured by di|uting
samples with Isoton (Coulter Electronics), ag/ta~ng
diluted samples in an MSE homogenizer to separate
clumped cells and counting in an electronic particle
counter (Coulter Electronics). Cell growtii was also
measured by the increase in absorbanee at 450 am
using a spectrophotometer. Mean ceh size was determined using the electronic particle counter.
2.3. Synchronous cultures
2. Materialsand Methods
2.1.Strains and growth conditions
Haploid strains 2180-1 b and A364~, and 1he temperature.sensitive mutants cdc7 (4008) and cdc31
(I~021) derived from A364a [2], wine used. The
batch culture media employed were l~,belled YEPD
(1% yeast extract, 2% bactopeptone, 2% de:~trose),
YEPG (1% yeast extract, 2% baczopeptone~ 2% glycerol), proline (0.2% proline, 2% dextrose, 0.14% yeast
nitrogen base without amino acids), and ethanol (2%
v/v ethyl alcohol (95%), 0.5% ammonium sulphate,
0.67% yeast nitrogen ba~e without amino acids). These
media also contained amino acid supplements, trace
Exponential cultures of 2180-I b in YEPG medium
were fractionated according to cell size by se~/mentation velocity centrifugation using a zonal rotor [4,5].
Fractions containing small unbudded cells were used
to inoculate a synchronous culture in fresh YEPG
medium at 24°C.
2. 4. Bud scar analysis
Cells were stained with the optical Orightener calcofluor. Cells were examined using a Zeiss fluorescence
microscope (fitted with UGI and BG38 primary filters
and a LC30 secondary fflteO. Daughter ~|Is (sept,
rated buds), which were unbudded, posse.~sed no m~d
scars and budded daughters possessed a lluore,~em
2~6
bud scar at the site of bud initiation. Unbudded parept
cells posse~ed one or more bud scars, and budded
parents possessed two or more bud scars. The perce,tage of daughters and parents in the population was
determined.
3, Results and Discumon
A variation of mean cell size with growth rate was
o,~served when cells were grown in batch culture
Gable 1); th~ faster the growth rate the larger the
cells. At fast growth rates it is a common observation
that the pareltt cell (the cell that produces the bud)
and the daughter cell (the bud) are equal in size at cell
separa.'ion. The observed decrease in mean size at slow
growlS, rates could arise if daughters at cell division
were smaller than the parent cell. In this case small
daughter cells might require a period of time to grow
to the size of a parent cell before themselves producing
a bud. Cells once having reached parent size might
produce buds at regular intervals without appreciable
further growth. [his model of yeast growth predicts
that at fast growth rates parent cells and da,ghter cells
should have the same generation times whereas at slow
growth rates the generation time of daughters should
oe longer than that of parents.
If the generation times of parents and daughters are
~he same then an exponential culture should contain
50% daughters. If however the generation time of
daugitters is longer than parents, the proportion of
daut0tters should be greater than 50%. The reason for
this i; that, at division, parents produce a parent and
a daughter (a net gala of one daughter), l~ daughter
c~U at division becomes a parent cell and produces a
daughter (a net gain of a parent). If parents divide
falter than ,'aughters, they produce more daughters
than daughten produce parents and thus the percent.
ago of daughters in the population is larger than 50%.
Cells were grown at different rates and the percent.
age of daughters was determined at etch growth ~,ate.
'l~e results, shown in Table 2 indicate that the per.
ce,lta~e daughters increased with decre~ng growth
rate in both batch and chemostat cultures, as expected
if daughters have a longer generation time than parents.
The discrepancy of generation times of daughters and
parents increased as growth rate decreased.
When synchronous cultures of yeast are made at
fast growth rates cell numbers in the cultltre double
synchronously, and some time later double again such
that cell numbers inclease from N to 2N 'and from 2N
TABLE I
Mean cell volume at various mass doubling time~in batch culture
Strain
Medium
Mass doubling
time (It)
Mean cell volume O~m3)
A364a
4001~
A36,lt
4008
A364a
4008
A364a
4008
YEPD
YEPD
I~oline
~oline
YEPG
2.83
3.00
3.67
3.67
4.92
4.75
7.33
7.]7
50.40
49.14
40,22
42.81
30.24
32.75
26.46
28,98
YEPG
Ethanol
Ethanol
TAF:IE 2
Per<enrage daughters at variousgrowth rates
Sara n
Medium
Specific~owth
Massdoubllna
rate ( h - : )
time (h)
% daughters
% patents
218O-lb
2180.lb
2180-1b
2180-1b
YEPD
Proline
YEPG
Ethanol
0.23
0.208
0.160
0.108
3.0
3.33
4.33
5.42
48
54
59
61
52
46
41
39
120-" I
120;:I
12021
Chemostat
C'hemostat
Chemosta,
0,26
0.16
0.055
2.67
4.33
! 2.6
55
59
64
45
41
36
237
to 4N, If a slow growing culture is composed of parents with relatively short generation times and daughters With relatively long generation times then e synchronous culture in media capable of supporting only
a slow growth rate would increase in cell number from
IN to 2N in the first division and 2N to 3N rather
than 4N in *,.hesecond. This result is expected if, at
the first division, the products of that division (viz.
parent and daughter) have different generation times,
and the parent is ready to produce another bud whereas the doughty, requires extensive growth to reach the
required size for bud initiation. The results of such an
experiment are shown in Fig. 1. An exponential culture grown on YEPG media was fractionated according to size, and unbudded cells of similar size were
used to initiate synchronous growth in fresh YEPG
medium. Cell numbers increased from 25 to 45 at the
first division and from 45 to 65 at the second. This is
equivalent to an increase from N to 2N and 2N to 3N.
This result was brought about by parents dividing for
_/f
a second time while ttle daughters, the product~ of the
first division, develop further. Indeed we have observed, in ethanol-corttaining media, that parents d:~de
yet again before division of the daughters prc<l~zc~
by the first division.
These results are compatible with the v,~ew that
yeast cells require to be of a certain size before they
can initiate a particular cell cycle event, pos~b[y, but
not necessarily, budding. It has been prop~J~ed that
the step identified by the cdc28 mutation is "start"
in the cycle [6], and perhaps co~npletion of this ev¢~,"
is dependent on the attainment of a ctith:al cell ~.c.
I f a critical size is necessary for completion of a
cell cycle event, close in time to bud initiation, aa~ ~f
once this event is completed cells proceed to call d~dsion after a period of time which is indelxndent of
growth rate, then while the bud can reach the size ¢f
the parent in fast growing cultures, in slow growing
cultures the bud cannot attain the ~ of the parent
at the time of cell sgparati(~n. Bud size at ceU separation will be inversely proportional to the mass doubling of the culture, which is compatible with the
results shown in Table 2.
Attainment of a critica~ ceil size before a c o r n p ~
tion of a cell cycle event can occur provides a means
of coordinating growth and cell division.
Acknowledgements
We thank L.H. Hartwell and the Berkeley Stock
Center for yeast strains and E. Gilmartin for exce~]em
technical assistance. This study was supported by the
Medical Research Council of Ireland and A. Gumaess,
Son and Co. Ltd. M.N. Jagadish thanks foe Royal
Commission for the Exhibition of 1851 for a schc~.a~ship.
References
|
i,
200
i
_t.,,
400
~
i
J
600
MINUTES
Fig, 1, A synchronous culture of 8aecharomyces cerevista¢
i~owlng in YEPG medium. An exponential culrJze of straia
2180-1b was sea,stated Into fractions ~.ccordingto size. Fractions containing small unbudded cells were used to inoculs*.e
a synehronuus culture growing in ¥EPG medium, Cell numbers in the culture were determined at intervals.
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