J. Cell Set. 79, 247-257 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
247
WATER ORDERING DURING THE CELL CYCLE:
NUCLEAR MAGNETIC RESONANCE STUDIES OF
THE SEA-URCHIN EGG
S. ZIMMERMAN*
Division of Natural Sciences, Glendon College, York University, Toronto,
Ontario M4N 3M6, Canada
A. M. ZIMMERMAN
Department of Zoology, University of Toronto, Toronto, Ontario M5S 1A1, Canada
G.D.FULLERTON
Department of Radiology, The University of Texas Health Science Center at San
Antonio, San Antonio, Texas, U.SA.
R. F. LUDUENA
Department of Biochemistry, The University of Texas Health Science Center at San
Antonio, San Antonio, Texas, U.SA.
AND I. L. CAMERON
Department of Cellular and Structural Biology, The University of Texas Health Science
Center at San Antonio, San Antonio, Texas, U.SA.
SUMMARY
Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T\)
during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following
insemination there was a 90% increase in the T\ value. The increase in T\ at fertilization could be
accounted for by the accumulation of extracellular fluid between the egg surface and the
fertilization envelope. The T\ value then remained without change during the first cell cycle, except
at metaphase when there was a significant 13% decrease. The lowered T\ values measured at
metaphase were not related to a change in the water content of the packed cells, which remained
fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and
colchicine (agents that depolymerize mitotic apparatus microrubules) did not affect the T\ values in
fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and
colchicine resulted in an increased T\, which accompanied the depolymerization of microtubule
protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high
pressure, colchicine or low temperature does not alter the T\ of water protons in the cell, it is
proposed that the increased state of ordered water molecules at metaphase is maintained by nonmicrotubular factor(s) of the metaphase egg.
INTRODUCTION
Nuclear magnetic resonance (n.m.r.) spectroscopy is a non-invasive method that
permits study of the physical properties of water molecules in cells, in particular their
capacity for motional freedom. Measurements of the spin-lattice relaxation time (T{)
•Author for correspondence.
Key words: cell cycle, nuclear magnetic resonance, sea-urchin eggs.
248
5. Zimmerman and others
reflect the freedom of water after perturbations by an electromagnetic pulse. Longer
T\ values are associated with freedom of movement and less organized water,
whereas shorter T\ values are a reflection of less freedom of movement and a more
organized state of the water molecules. There has been extensive scientific study
of the physical properties of water in biological systems (see reviews by Ling,
1962; Drost-Hansen & Clegg, 1979). Beall and coworkers (Beall, Hazlewood & Rao,
1976; Beall, Chang & Hazlewood, 19786; Beall, 1980; Beall, Brinkley, Chang &
Hazlewood, 1982) have investigated the state of water in HeLa, Chinese hamster
ovary (CHO) and breast cancer cells. Their findings indicate changes in the motional
freedom of water associated with the cell cycle and with the extent of assembly of
micro tubules in normal and in cancer cells. In this study, we investigated water
ordering during the cell cycle of sea-urchin eggs. On the basis of past reports we
anticipated changes in T\ at the time of assembly of microtubules in the mitotic
apparatus. A preliminary report of this study was published elsewhere (Zimmerman
etal. 1984).
MATERIALS AND METHODS
Organisms
Sea urchins, Strongylocentrvtus purpuratus, were purchased from Pacific BioMarine Laboratories, Venice, California. Eggs were obtained by intracoelomic injection of 0-53M-KC1. The
shed eggs were washed three times with 250 ml artificial sea water (Instant Ocean) before use.
Sperm were obtained from excised testes and stored at 4°C. Diluted sperm suspensions were
freshly prepared before use. Only those batches of eggs in which at teast 98% of the eggs showed
fertilization membranes within 5 min of insemination were used for experimental purposes. After
fertilization the zygotes were placed in sea water at 18 °C, at which temperature cells go through
cytokinesis 90-110 min after insemination. Studies by Fry (1936) and Harvey (1956) indicate that
the time of appearance of mitotic events is fairly constant during the first cleavage cycle. Thus it is
possible to relate cleavage cycles from various batches of eggs. On the basis of previous experience,
the division times for different batches of eggs were standardized at 100 min.
Nuclear magnetic resonance (n.m.r.) measurements
A 400-500 (A sample of concentrated cells was placed in an n.m.r. capillary tube and uniformly
packed by centrifugal force (272^for 3 min at room temperature). It was determined by packed cell
volume 'haematocrit' measurements that unfertilized and fertilized sea-urchin eggs attain uniform
packing volume in the capillary tube after 2 min of centrifugation (at 272 g), after which the
fertilization envelopes of adjacent eggs were packed into a hexagonal pattern, indicating that little if
any space remained outside the fertilization envelope. There was no further measurable change in
cellular packing after longer periods of centrifugation. Pulsed proton n.m.r. relaxation measurements were made on separate equal samples of packed eggs at different times in the first cell cycle
using a Praxis model II instrument (San Antonio). This instrument has a 0-25 tesla permanent
magnet, sample coil and RF pulser tuned to 10-7 MHz. The instrument is interfaced with a
microcomputer for fast data acquisition and has built in data analysis software. The T\ or spinlattice relaxation time was measured using the saturation recovery pulse sequence, 90°—T-90°. The
resultant analysis of 30 free-induction-decay peak heights with a sequence of increasing interpulse
delay times yields the Tl decay curve presented in Fig. 1. Such uni-exponential decay curves
permitted the determination of the relaxation time from the least-squares fit to all 30 data points.
Water content and cell diameter measurements
Following the n.m.r. measurements the individual samples were removed from the n.m.r. tube,
weighed in pre-tared weighing pans and then dehydrated in a vacuum oven at 90 °C over a period of
n.tn.r. studies of sea-urchin eggs
249
several days until a stable weight was achieved. The difference between the initial wet weight of the
samples and the final dry weight was used to determine the percentage of water in the samples. A
calibrated ocular micrometer was used to measure the diameter of the egg proper, both before and
after fertilization. The diameter of the raised fertilization envelope after fertilization was also
measured. The volume of the egg proper as well as the volume of the space between the egg and the
raised fertilization envelope could be calculated from the diameter measurements because of the
spherical shapes.
Pressure experiments
n.m.r. capillary tubes containing 400-500/11 of packed cells or experimental solution
(microtubule protein solution) were filled with paraffin oil and covered with a soft plastic cap. The
n.m.r. tubes were placed in a stainless steel pressure chamber. The pressure chamber was
connected to a hydrostatic pressure pump (Zimmerman, 1970). Hydrostatic pressures up to
l-6xl0 4 lbf in" 2 (equivalent to l - l O x ^ k N m " 2 ) were applied with an Amico pressure pump at
the rate of 5 X103 lbf in" 2 (3 -45 X104 kN m~2) per stroke at a temperature of 23 C C. Decompression
was achieved almost instantaneously by means of a needle valve. About 60 s were required from the
time of decompression to the start of the n.m.r. measurements.
Tubulin
Microtubule protein was prepared from rat brain by a cycle of assembly and disassembly using
the method of Fellous, Francon, Lennon & Nunez (1977). The microtubule protein is obtained
in 100mM-2-(iV-morpholino)ethanesulphonic acid, pH6-4, 1 mM-ethyleneglycolbis(/3-aminoethylether)-N, W-tetraacetic acid, 0-1 mM-ethylenediaminetetraacetic acid, 0-5 mM-MgClz, 1 mMG T P and 1 mM-^-mercaptoethanol. The isolated protein contains about 65-70% tubulin and
about 20% microtubule-associated proteins. The microtubule protein is temperature-sensitive. At
0°C the protein is clear and in a fluid state. At room temperature it is cloudy and gel-like
(polymerized). The polymerization reaction can be reversed by lowering the temperature to 0°C.
RESULTS
Nuclear magnetic resonance spectroscopy was used to monitor the freedom of
motion of water molecules in sea-urchin eggs, 5. purpuratus. Individual samples of
the sea-urchin eggs were uniformly packed into n.m.r. glass sample tubes and spinlattice relaxation times (T{) were measured in unfertilized eggs and fertilized eggs at
specific stages following insemination, i.e. syngamy, interphase, metaphase and
division. Cells removed from n.m.r. tubes after cell packing showed a division
schedule like that of the control cells. This observation indicates that packing of the
eggs had no adverse effect on their progression through the cell cycle. Since T\ values
relate to mobility of water molecules, these measurements may be influenced by
macromolecular structural changes as well as changes in water content of cells.
Therefore, it was necessary to determine the water content of the sea-urchin eggs at
all the developmental stages studied, since changes in water content could alter T\
values. Although the percentage hydration was lower in the unfertilized eggs, no
statistically significant differences were found in the water content of unfertilized
eggs compared with fertilized eggs, throughout the first cell cycle (Fig. 2B).
T I values at fertilization and during the first cell cycle
In general, the mean water proton relaxation time of unfertilized sea-urchin eggs
(520 ms) was significantly shorter than that of fertilized sea-urchin eggs at each stage
250
S. Zimmerman and others
of the cell cycle measured. There was a 90% increase in mean T\ (991 ms) 15 min
after insemination (syngamy), which remained fairly constant throughout the cell
cycle except at metaphase. The mean diameter of 24 unfertilized eggs proper was
76±0-5/im, yielding a calculated volume of 2-30X105 jun3, which remained the
same after fertilization. The mean diameter of the raised fertilization envelope was
100-8 ± 0-4/im, yielding a calculated volume of space between the egg and fertilization envelope of 3-O6xlOs/«n3. At metaphase, the mean T\ value was 896ms;
this represents a 13% decrease from the interphase value (1024 ms). The shorter T{
value at metaphase represented a significant change from the other T\ values during
the cell cycle. At cell division,T\ returned to the pre-metaphase value (Fig. 2A). The
more rapid water relaxation time at metaphase was not due to a change in water
content at this mitotic stage but may relate to assembly of the mitotic apparatus. In
particular, we thought that polymerization of tubulin into spindle microtubules
associated with the mitotic apparatus might be responsible for the decrease in water
proton relaxation time. To test this hypothesis, sea-urchin embryos were treated
with various spindle microtubule depolymerizing agents (hydrostatic pressure, low
temperature and colchicine) and T\ values were measured.
Influence of hydrostatic pressure on T\ values
Fertilized eggs, at specific cell cycle stages were uniformly packed in n.m.r.
capillary tubes and treated with hydrostatic pressure of 10000 lbf in" 2 for 1 min at
3000
2000
1000
-r
300
600
Tune (ms)
900
Fig. 1. A representative T\ decay curve of sea-urchin eggs at metaphase. A longitudinal
proton (Ti) relaxation decay curve obtained using a 90°-r-90° pulsed sequence on the
Praxis model II pulsed n.m.r. analyser with a 0-25 tesla permanent magnet sample coil
and RF pulser tuned to 10-7MHz. The amplitude intercept is 3258; Tu 722ms;
correlation coefficient, 0-997.
n.m.r. studies of sea-urchin eggs
251
1100
f 90°
700 -
/
i
7 Unfertilized
500
20
100
40
60
80
100
B
I
* 90
^Unfertilized
Syngamy
80
Metaphase
i
20
40
60
80
Time after insemination (min)
Division
i
100
Fig. 2. Water proton T\ and the % of water in eggs of the sea urchin, 5. purpuratus, are
shown at various stages of the cell cycle. Each measurement was made on a separate
freshly packed sample of eggs. The Tx at metaphase is statistically lower than the other Tx
values in the fertilized eggs. The water content of the fertilized and unfertilized eggs did
not change at each of the stages.
23 °C. This pressure and duration of treatment are known to depolymerize spindle
microtubules (Zimmerman & Marsland, 1964; Zimmerman, 1970; Salmon, 19756).
Following decompression, Tj values were determined. There were no statistically
significant differences between the Tx values for control and pressure-treated cells at
syngamy, interphase, metaphase or division (Table 1). Pressure-treated cells were
removed from the n.m.r. tubes and observed. The division schedule of these cells
was delayed (cf. Zimmerman & Silberman, 1965).
Low-temperature effects on Ti
Cells at metaphase were placed at 0°C in an ice-water bath for 20min; the cells
were returned to room temperature following which Tx measurements were made.
Low temperature is known to depolymerize spindle microtubules (Inoud, 1964).
Measurements of mean T\ values for low temperature-treated cells (800 ± 32) and
control cells (781 ± 23) showed no statistically significant differences.
252
5. Zimmerman and others
Effects ofcolchicine on T]
Fertilized eggs at early metaphase were placed into 2-5xlO~4M-colchicine for
40-54 min. This concentration of colchicine depolymerizes spindle microtubules (cf.
Sauaia & Mazia, 1961; Zimmerman & Zimmerman, 1967). The mean 7^ value for
the colchicine-treated cells (863 ± 62) was not statistically different from that for
non-treated control cells (781 ± 23). The water content of colchicine-treated eggs at
metaphase was 88-00 ± 1-25%; this value was similar to that for metaphase control
cells (89-25 ±1-39%).
Effects ofphysical and chemical agents on Ti in vitro tubulin studies
To test further whether polymerized and depolymerized states of tubulin reflect
differences in water proton T\ values, similar depolymerization agents were used in
studies in vitro with microtubule protein samples prepared according to the method
of Fellous et al. (1977).
The polymerized state of the microtubule protein preparation (17-13 mgml" 1 ) in
the presence of GTP and Mg2"1" is temperature-dependent. At 23 °C, microtubule
preparations appeared cloudy and gel-like (Fig. 3), indicating the polymerized form
of tubulin. Glass beads placed on the surface of this material did not fall through the
solution at unit gravity. When this microtubule protein sample was put in an ice bath
at 0°C, it became clear and fluid-like (depolymerized) within 5 min. Glass beads
placed on the surface of this preparation fell rapidly through the solution. The
correlation between temperature and the state of polymerization of the microtubule
protein preparation was further borne out by n.m.r. measurements of the spin-lattice
relaxation time for water molecules in this material. The mean T\ value at 23 °C was
Table 1. Pulsed proton Ti values (ms ± s.E.M.) offertilized sea-urchin eggs during
the first cell cycle and immediately after exposure to high hydrostatic pressure (1 min
at 10000 Ibf in'2)
Time after
fertilization
(min)
15
53
75
100
Stage of the
cell cycle
Syngamy
Interphase
Metaphase
Division
Treatment conditions
n
No pressure
8
991 ±99
1024 ± 35
896±60
1043 ±109
5
9
5
Row mean
975
n High pressure
8
5
9
5
1027 ± 77
1004 ± 6 8
933 ± 45
1089 ±104
1003
Column
mean
1009
1014
914*
1066
Results of a two-way analysis of variance test:
Stage of cell cycle
Treatment condition
Interaction
F value
10-751
1-932
0-444
P value
< 0-001*
Not significant
Not significant
•Results of Student-Newman-Keul's multiple-range test showed that the value at metaphase
was significantly different from all other column mean values at P< 0-0023; none of the other
column mean values were significantly different.
n.m.r. studies of sea-urchin eggs
253
Fig. 3. Photographs showing polymerized (A) and non-polymerized (B) microtubule
protein preparation (1-7%). The opacity of the polymerized sample (at 23 °C) is evident
when compared with the non-polymerized (0°C) sample.
1316 ± 16-9ms, whereas at 0°C it was 1511 ±5-9ms; these values are statistically
different (P< 0-01). The increase in relaxation time reflects the greater freedom of
mobility of water molecules that exists in the depolymerized state.
Colchicine, at concentrations of 2-5XlCT4M and 2-5xlO~6M, was added to the
microtubule protein preparation at 0 °C and the preparation was allowed to warm to
23 °C. The-change in temperature from 0°C to 23 °C occurred within 5min. The
mean Tx values in the presence of 2-5xlO~4M and 2-5xlO~6M-colchicine were
1539 ±18-3 and 1456 ± 16-8 ms, respectively (Fig. 4). These 7\ values for the
colchicine preparations were statistically different (P<0 - 01) from that of control
microtubule protein at 23°C (1316± 16-9ms) (Fig. 4). Moreover, the T\ values for
the two colchicine-treated preparations were statistically different from one another
(P<0-01). At thermal equilibrium (23 °C), 10-15 min after the preparations were
removed from 0°C, the difference between T\ values for colchicine and control
samples exceeded 100 ms.
Another depolymerizing treatment applied to microtubule protein preparations
was hydrostatic pressure of 160001bf in" 2 for 30 min at 23 °C. n.m.r. measurements
of Tx were initiated 60 s after decompression. The mean Tj was 1348 ± 6-4 ms, which
was not statistically different from the mean T\ of non-pressurized microtubule
protein at 23 °C (Fig. 4). Since there was the possibility of a rapid reversal, from the
depolymerized to the polymerized state, upon decompression, glass beads were
placed on the surface of the polymerized microtubules before compression.
Observations of these experiments revealed that some of the glass beads had moved
down about 0-5 cm below the surface of the material, which may indicate a partial
depolymerization effect.
254
5. Zimmerman and others
1600 -
1200
Treatment
Temperature
16000
lbfin"2 colchitine
—
0°C
23°C
23°C
23°C
colchicine
23°C
Microtubule protein
Fig. 4. Effect of low temperature, hydrostatic pressure and colchicine treatment on the
T\ of microtubule protein preparations (Yl^XZugmT^). At 0°C the microtubule
preparation was clear and in a fluid state (depolymerized). When the tubulin preparation
was warmed to room temperature it became cloudy and changed to a gel state
(polymerized). This polymerized state is reflected in a shortening of the 7\. Microtubule
preparations at 23 °C subjected to pressures of 160001bf in" 2 for 30min showed no
change in T\. Tubulin preparations at 23 CC treated with colchicine at concentrations of
2-SxlO~ 4 M and 10~6M showed increases in T\ that were directly proportional to the
colchicine concentration.
DISCUSSION
What accounts for the 90 % increase in the mean water proton relaxation time upon
fertilization? It seems reasonable to think that the longer T\ value for fertilized eggs
might be due to the increased fluid in the space between the egg membrane and the
raised fertilization envelope, which was not removed by packing of the fertilized
eggs. Assuming a model of fast proton exchange between the egg and this space in the
packed eggs we can evaluate this suggestion using the following equation:
l/Ti=/.xl/TIa+/bXl/7'Ib>
where l/Tj is the relaxation rate of the packed fertilized eggs, / a is the volume
fraction of the space between the egg membrane and the fertilization envelope (0-57),
l/^ia is the relaxation rate of the fluid in this space (assuming it is sea water, this
value is 0-37), / b is the volume fraction of the egg proper (0-43), l/Ti b is the
n.m.r. studies of sea-urchin eggs
255
relaxation rate of the egg proper (because we could not measure this value in the
fertilized egg we have assumed it is the same or similar to the value of the unfertilized
egg, 0-0019). Solving for T\ we obtain 988ms as the expected 7\ value. This
expected value compares with an observed measured value of 991 ms for the newly
fertilized eggs. Thus the increase in relaxation time at fertilization can be accounted
for by the increase in the fluid in the space between the egg surface and the raised
fertilization envelope.
These studies show a significant decrease in the T\ values for water molecules in
fertilized sea-urchin eggs during metaphase compared with other stages of the cell
cycle. The lower T\ values measured at metaphase were not related to a change in
water content of the fertilized eggs, which remained fairly constant throughout the
cell cycle. One possibility for the reduced Tx value seen at metaphase may be related
to the assembly of a mitotic apparatus, whose macromolecular structure may cause a
greater ordering of water in the cell. The mitotic apparatus, formed at metaphase, is
an extensive structure that comprises approximately 12% of the total cell protein
(Mazia & Roslansky, 1956). Thus water molecules may become increasingly
associated with this macromolecular structure, thereby reducing their freedom of
motion. Other possibilities for the change in T\ at metaphase also exist.
The pattern of Tj values during the cell cycle in sea-urchin eggs is different
from that of HeLa and CHO cells (Beall et al. 1976; Beall, Bohmfalk, Fuller &
Hazlewood, 1978a; Beall, 1980). n.m.r. measurements during the cell cycle in
synchronized HeLa and CHO cells showed the longest T\ times during mitosis and
the shortest Tj times in the S phase. The percentage hydration of these cells was
higher in mitosis and lower in 5 phase. Nevertheless, Beall (1980) states that the
differences in T\ in mitosis and 5 phase in HeLa cells cannot be attributed entirely to
the change in water content since T\ values increased from 5 to Gi with no change in
water content occurring at that time. Beall and coworkers (1976) relate the
differences in T\ values to the chromatin condensation cycle of the cell and proposed
a greater binding of water to diffuse chromatin than to chromatin in condensed
chromosomes. The relationship of water-ordered structure to specific cell cycle
stages in the sea urchin is the opposite to that in HeLa and CHO cells. In sea-urchin
eggs chromatin may have less involvement in water ordering since there is a large
cytoplasmic to nuclear ratio during the cleavage period, as opposed to a larger nuclear
to cytoplasmic ratio in HeLa cells (Beall, 1980).
Beall et al. (1982) suggested that the polymerization and depolymerization of
tubulin in cells is correlated with the behaviour of cell water. They reported increases
in T\ values in human breast cancer cells with less microtubule material and
decreases in T\ in cells with more microtubule material. In addition, they found that
T\ increased in HeLa and CHO cells treated with the depolymerizing agent,
colcemid. Thus we reasoned that the microtubule complex associated with the
mitotic apparatus, in sea-urchin eggs at metaphase, might play an important role in
the decrease in Tx observed at this cell-cycle stage. However, sea-urchin cells treated
with depolymerizing agents such as hydrostatic pressure, low temperature and
colchicine did not show any significant changes in the motional freedom of water
256
S. Zimmerman and others
molecules. When microtubule protein preparations were treated in vitro with these
depolymerizing agents, the Tl values increased significantly, except in the case of
hydrostatic pressure, where there was no change in T\ measurements. The absence
of a measurable change in T\ as a result of pressure treatment was unexpected, since
hydrostatic pressure is known to depolymerize microtubules in vivo (Tilney,
Hiramoto & Marsland, 1966; Kennedy & Zimmerman, 1970) andm vitro (Salmon,
1975a). Since pressure effects are rapidly reversible, the microtubule protein
solution may have repolymerized before n.m.r. measurements could be made.
The in vitro experiments clearly show that microtubules can influence the ordering
of water. Nevertheless, the depolymerization of microtubules by high pressure, low
temperature and colchicine, in sea-urchin eggs, was not reflected in the T\ values
of the water molecules in these cells. Thus the significant shortening of the water
proton T\ seen at the metaphase stage of the cell cycle is not due to decreased water
content of the cell nor to the known increase in microtubule assembly. We propose
that the ordered state of water molecules at metaphase is maintained by other
macromolecular structures of the metaphase cell, which are not disrupted by
depolymerization of the microtubules.
This work was supported in part by grants from the USPHS (no. CA36372) to I.L.C., from the
NSERC to A.M.Z., by Glendon College Research grants to S.Z., and a USPHS grant (no.
CA26376) to R.F.L. This work waa carried out in the Department of Cellular and Structural
Biology, The University of Texas Health Science Center at San Antonio while S.Z. and A.M.Z.
were visiting scientists. The authors thank Loree Cameron for her support and Veena Prasad for
her skilled technical assistance.
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