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Cell Motility and the Cytoskeleton 37:263–270 (1997)
Effects of Intracellular pH on the Mitotic
Apparatus and Mitotic Stage
in the Sand Dollar Egg
Kenji Watanabe, Miyako S. Hamaguchi, and Yukihisa Hamaguchi*
Biological Laboratory, Faculty of Bioscience and Biotechnology,
Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, Japan
The effect of change in intracellular pH (pHi) on mitosis was investigated in the
sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and
Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of
treating the egg at nuclear envelope breakdown with sea water containing acetate
and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became
larger than that of normal fertilized eggs; that is, the mitotic spindle had the
maximal size, especially in length at pHi 6.70. The spindle length linearly
decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the
increase in birefringence retardation was detected at slightly acidic pHi, suggesting
that the increase in size of the spindle is caused by the increase in the amount of
microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus
was inhibited.
Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By
counting the number of the eggs at various mitotic stages with time after treating
them with the media, it is found that metaphase was persistent and most of the S.
mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is
concluded that at slightly acidic pH, the microtubules in the spindle are stabilized
and more microtubules assembled than those in the normal eggs. Cell Motil.
Cytoskeleton 37:263–270, 1997. r 1997 Wiley-Liss, Inc.
Key Words: acetate; ammonia; birefringence; cleavage; microtubule; spindle
During cell division, the mitotic apparatus plays a
role in separating daughter chromosomes into two groups
and transporting the cleavage stimulus to the cell cortex at
the equator. In higher eukaryotes, the mitotic apparatus is
organized shortly after nuclear envelope breakdown
(NEBD). Its morphology changes dynamically through
mitosis, and this change is induced by assembly and
disassembly of microtubules. It is known that there are
many factors affecting the assembly and disassembly by
means of studying them both in vitro and in vivo [see as a
review, Dustin, 1984]. Among these factors, pH is one of
candidates for controlling mitosis [Rozengurt, 1986; Epel
and Dube, 1987] and it is reported that intracellular pH
(pHi) changes occurred during cell division in the frog,
Xenopus and in the slime mould, Physarum [Gerson and
r 1997 Wiley-Liss, Inc.
Burton, 1976; Lee and Steinhardt, 1981; Webb and
Nuccitelli, 1981; Grandin and Charbonneau, 1990]. In the
sea urchin egg, pHi rises at fertilization by 0.5, which is
necessary for further development. Shortly after the
increase in pHi, the sperm aster forms, syngamy occurs,
and then the progression for the first cleavage starts
[Hamaguchi, 1982]. Isolation of the mitotic apparatus of
Kenji Watanabe’s current address is Laboratory of Symbiosis and
Transmission, Department of Insect Physiology and Behavior, National
Institute of Sericultural and Entomological Science, Ohwashi, Tsukuba,
Ibaraki 305.
*Correspondence to: Yukihisa Hamaguchi, Biological Laboratory,
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152, Japan.
Received 14 January 1997; accepted 3 March 1997
Watanabe et al.
the sea urchin egg was carried out using various media at
a wide range of pH values and it is found that slightly
acidic pH is adequate for the isolation [Kane, 1962].
However, the effect of pH on the mitotic apparatus is not
known sufficiently in the living cell.
It is known that pHi can change by the treatment
with a medium containing acetate or ammonia and this
has been widely used [for a review, Roos and Boron,
1981]. The principle of this method is briefly as follows.
When the pH of a medium containing acetic acid as weak
acid is lowered, molecules of acetic acid but not acetic
ions produced in the medium can permeate the cell
membrane. Inside the cell, they are dissociated into H1
and acetic ions, which results in the decrease in pHi. On
the contrary, when the pH of a medium containing
ammonia is elevated, molecules of ammonia enter the
cell-associated H1, and become ammonium ions. As a
result, pHi is shifted higher [Jacobs, 1940]. In sea urchin,
this method has been utilized in many investigations
[Keller et al., 1980; Shen and Steinhardt, 1980; Schatten
et al., 1985; Dube and Epel, 1986]. Recently we have
developed the method of controlling pHi precisely under
physiological conditions [Hamaguchi et al., 1997].
In this study, in order to reveal the dynamic nature
of the mitotic apparatus during cell division, we have
investigated the effect of pHi changes on mitosis, especially focusing on the morphology of the mitotic apparatus and the progression of the mitotic stages. We found
that at slightly acidic pH, the mitotic apparatus became
larger and stabilized, and the mitotic stage was arrested at
metaphase but that the mitotic spindle decreased with
alkaline pHi.
Biological Materials
Gametes of sand dollars, Scaphechinus mirabilis
and Clypeaster japonicus, were used as materials. Gametes were obtained by injection of 1 mM acetylcholine
dissolved in sea water into the body cavity. Eggs were
washed three times by artificial sea water (Jamarin-U,
Jamarin Laboratory, Osaka, Japan), kept at 15°C, and
used within 4 hours of shedding. Sperm were collected
‘‘dry,’’ kept at 4°C in a refrigerator and diluted just before
use. Fertilized eggs were deprived of both the fertilization
envelope and hyaline layer by treating them with 1 M
urea solution for 1.5 min shortly after insemination and
incubated in Ca free sea water (Ca-free Jamarin-U,
Jamarin Lab) at 20°C and 25°C in the case of S. mirabilis
and C. japonicus, respectively. The fertilized eggs were
used for pHi measurements and treated at the onset of
NEBD with sea water containing ammonia and/or acetate
at various pH values (pHSW). Several minutes after the
treatment, they were permeabilized and extracted with a
TABLE I. The Composition of pHSW (mM)
pH range
10, 20
426, 416
10, 20
426, 416
shows the same figure as the left column. pH was adjusted by
microtubule-stabilizing solution (10 mM EGTA, 25 mM
MES (2-morpholinoethanesulfonic acid), 1 mM MgCl2,
25% glycerol, 1% Nonidet P-40; pH was adjusted to 6.7
by KOH). These eggs were used for morphological and
birefringence measurements directly or after immunofluorescence staining.
Sea Water
The compositions of various kinds of pHSW used
in this study are summarized in Table I. Briefly, AcpHSW, NH3-pHSW and Ac1NH3-pHSW were Ca free
sea water (CaFSW) containing acetate, ammonia, and
both acetate and ammonia, respectively, and pH was
changed from 6.2 to 8.0. Normal pHSW was normal
CaFSW, just containing PIPES (1,4-piperazinediethanesulfonic acid) or HEPES (N-2-hydroxyethylpiperazine-N83-propanesulfonic acid) whose pH was adjusted to 6.2–
8.0. The eggs of S. mirabilis were treated with Ac-pHSW,
or NH3-pHSW containing 20 mM of acetate and/or
ammonia at various values of pH, whereas the eggs of C.
japonicus were treated with those containing 10 mM of
acetate and/or ammonia.
pH Measurements
pHi was measured using the fluorescent pH indicator dye, pyranine (1-hydroxypyrene-3,6,8-trisulfate, Tokyo Kasei Kogyo Ltd., Tokyo, Japan). Pyranine solution
at 10 mM in 50 mM MOPS (3-(N-morpholino) propane
sulfonic acid, pH 7.0) was injected at 0.3–0.4% of the egg
volume (final concentration is 30–40 µM in the egg
cytoplasm) into the fertilized egg which was attached to
the polylysine-coated coverslip by the method as described by Hiramoto [1974] and Hamaguchi [1982]. In
order to treat the eggs with the media, the pHSW was
applied to the one edge using a pipette and absorbed with
a piece of tissue paper from the other edge.
Fluorescence intensities of the egg were measured
before and after perfusing various pHSW, using a Nikon
photomicroscope equipped with epi-illuminating appara-
Effects of pHi on Mitosis in Sand Dollar Egg
tus (EFD, Nikon). Light passed through one of two
interchangeable excitation filters (415 or 454 nm narrow
band pass interference filter, Nippon Sinku Kogaku,
Tokyo, Japan) illuminated the sample through an objective (Fluor 20x/ NA 0.75). Fluorescence was collected by
the objective and passed through the dichroic mirror and
then through a barrier filter (.520 nm). It was then
detected with a photomultiplier tube. The signal was
plotted with a chart recorder. The ratio of fluorescence
intensity excited at 454 nm to that excited at 415 nm was
pH calibration was carried out in the pH reference
solutions supplemented with 30 µM of pyranine. The
composition of the solutions was as follows, 40 mM
MES, 40 mM MOPS, 40 mM EPPS (3[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid) and 106–175
mM KCl (pH 6.3, 6.8, 7.3, or 8.0, the ionic strength 200
mM). Fluorescence intensities at two wavelengths of 454
and 415 nm were measured 3–6 times in the solutions
loaded into a microslide with 100 µm in depth. The
fluorescence ratios were calculated and a pH calibration
curve was made.
Indirect immunofluorescence was carried out using
anti-tubulin antibody as described earlier [Oka et al.,
1994]. The extracted fertilized eggs were attached to the
polylysine-coated coverslip and then fixed with 100%
cold methanol. They were stained with the antibody,
DM1A (Amersham, England) for microtubule observation and then stained at 0.1 µg/ml with DAPI (48,6diamidino-2-phenylindole, Sigma Chemical Co., St. Louis,
MO) for chromosome observation.
Measurements of the Spindle Length and Width,
and Determination of the Mitotic Stage
The mitotic stage of the cell was determined by
observing chromosomes with fluorescence and DIC microscopes and the mitotic stages of the cells were divided
into five groups of cells at prophase, prometaphase,
metaphase, anaphase, and telophase; populations of the
cells at each stage were counted and then summarized
into three groups at prophase or prometaphase, metaphase, and anaphase or telophase. The spindle length
(pole to pole length, L) and spindle width (W) of the
mitotic apparatus at metaphase were also measured.
Calculation of Birefringence Retardation and
Microtubule Number in the Mitotic Spindle
The extinction angle (z) of the mitotic spindle in the
extracted cell was measured with a polarization microscope equipped with a rectified objective (40x/ NA 0.65)
and a Brace-Kohler compensator (retardation [R0] of
27.7 or 29.0 nm) and then birefringence retardation [R] of
Fig. 1. Time courses of pHi change after the treatment with pHSW.
The eggs were treated with NH3-pHSW at pHo 8.0 (squares), with
Ac1NH3-pHSW at pHo 7.3 (triangles), and with Ac-pHSW at pHo 6.8
(circles). The abscissa is time after treatment with those pHSW, and the
ordinate is pHi.
TABLE II. The Relation Among pHo of pHSW and pHi Before
Treatment and pHi After Treatment
pHi before
7.34 6 0.02
7.31 6 0.09
7.43 6 0.07
7.35 6 0.13
7.37 6 0.09
7.30 6 0.11
7.39 6 0.08
7.42 6 0.08
pHi after
(10 min)
6.57 6 0.02
6.70 6 0.02
6.77 6 0.01
6.96 6 0.12
7.35 6 0.01
7.57 6 0.09
7.67 6 0.04
7.84 6 0.03
the spindle was calculated using the following equation
as described by Hiramoto et al. [1981a].
R 5 R0 3 sin (2 3 z)
The number of microtubules (MN) in the spindle
was then calculated using the following equations.
M 5 3.14 3 R 3 W/4
MN 5 2n2(n21 1 n22)M/(n21 2 n22)2A,
where M, A, n1, n2, and W are sum of the retardation over
the cross-section of the spindle, area of the cross-section
of a microtubule (3.0 3 10216 m2), refractive index of
microtubule (1.512), and refractive index of the medium
(1.335), and width of the spindle, respectively.
Watanabe et al.
Fig. 2. Immunofluorescence micrographs of the eggs at metaphase after the treatment with pHSW. A
control egg (a,b), the eggs treated with Ac-pHSW at pHo 6.2 (c,d), with Ac-pHSW at pHo 6.5 (e,f), with
Ac-pHSW at pHo 6.8 (g,h), with Ac1NH3-pHSW at pHo 7.3 (i,j ), and with NH3-pHSW at pHo 8.0 (k,l)
are shown. a, c, e, g, i, and k are immunofluorescence micrographs showing microtubules in the mitotic
apparatus, and b, d, f, h, j, and l are micrographs showing metaphase chromosomes stained with DAPI.
Bar 5 10 µm.
Fig. 3. The relation of spindle length and width in the egg treated with
pHSW containing acetate and/or ammonia at various pHo to the pHi.
The abscissa is pHi, and the left ordinate is spindle length (filled
circles) and the right ordinate is spindle width (open circles). Mean and
S.D. of the spindle length and spindle width in the eggs treated with
various pHSW are represented in percent to the spindle length and
width in the control eggs, respectively. S.D. is shown only positive or
negative value in the case of the spindle length or width, respectively.
Sample number is 15 to 22.
Fig. 4. Polarization micrographs of the mitotic apparatus. The mitotic
apparatus in the control egg (a) and that in the egg treated with
Ac-pHSW at pHo 6.8 (b) are shown after extraction. Bar 5 10 µm.
Effects of pHi on Mitosis in Sand Dollar Egg
TABLE III. Birefringence of the Mitotic Apparatus Treated With Ac-pHSW
Ac-pHSW (pHo 6.8)
Ac-pHSW (pHo 7.3)
Scaphechinus mirabilis
(3103 )
9.4 6 0.7
10.2 6 0.8
9.9 6 0.5
4.6 6 0.6
5.0 6 0.4
4.6 6 0.3
9.0 6 0.7
9.0 6 1.1
Clypeaster japonicus
(3103 )
4.4 6 0.2
5.0 6 0.5
is spindle width; R is birefringence retardation (nm); and MN is number of microtubules in the
cross-section of the mitotic spindle.
Morphological Change in the Mitotic Apparatus
Induced by the Change in pHi
Relationship between pHi and extracellular pH
(pHo). The pHi response of the fertilized egg of sand
dollars is summarized as follows. The pHi of the fertilized
egg was 7.34 6 0.10 (n 5 63) and 7.31 6 0.08 (n 5 15)
in S. mirabilis and C. japonicus, respectively. Typical
examples of time course of pHi change during treatment
with 20 mM pHSW containing acetate and/or ammonia at
various pHo (pH 6.8, 7.3, and 8.0) are shown in Figure 1.
When the eggs were treated with Ac-pHSW at various
values of pHo, the pHi decreased gradually and became
constant after 5–6 minutes of the treatment. On the other
hand, when the eggs were treated with NH3-pHSW and
NH31Ac-pHSW at various values of pHo, the pHi
increased to a maximal value transiently within 1 min
after the onset of application of pHSW, then decreased,
and became constant after 8–10 minutes of the treatment.
The values of pHi measured before the treatment and at
10 min of the treatment are summarized in Table II: pHi
of Ac-pHSW-treated eggs decreased, and that of NH3pHSW-treated eggs increased but that of NH31Ac-pHSWtreated eggs at pHo 7.3 did not change. The treatment
with normal pHSW at pH 6.2–8.0 did not induce any pHi
Change in the size of the mitotic spindle. In order
to examine the effect of pHi change on the morphology of
the mitotic apparatus, the eggs were extracted by a
microtubule-stabilizing medium after 8–10 min of the
treatment with various types of pHSW and stained with
anti-tubulin antibody. In the case of C. japonicus, the
eggs were treated for 7–11 minutes with those pHSW.
During this period, many eggs became metaphase. We
usually demonstrated the results obtained in S. mirabilis
because the extent of the effects obtained using S.
mirabilis was more remarkable than that using C. japonicus. The difference of the effect between two species may
be due to the fact that pHSW containing acetate and/or
ammonia in larger concentration was applied to S.
mirabilis than that to C. japonicus because the change in
pHi was estimated to decrease with its concentration in
the media.
As shown in Figure 2, various sizes of mitotic
apparatuses at metaphase were obtained by treating the
eggs with various types of pHSW. At pHi 6.70, the
mitotic apparatus appeared to be slender and became the
largest among all the mitotic apparatuses, and at pHi 7.84
it appeared short. At pHi 6.30, the mitotic apparatus was
poorly organized. The effects of pHi on the aster was not
great in comparison to that on the spindle. In these eggs,
the pole to pole length (L) and the width of the spindle
(W) of the mitotic apparatus were measured at metaphase. Those in the mitotic apparatus at metaphase in
control eggs are defined to be 100% (L 5 23.9 6 1.1 µm
and W 5 9.6 6 0.9 µm (n 5 100) in S. mirabilis and
L 5 23.3 6 1.6 µm and W 5 9.5 6 0.9 µm (n 5 137) in
C. japonicus). As shown in Figure 3, L was maximal at
pHi 6.70 and elongated by ca 20% in S. mirabilis.
Although W increased by ca 10% at pHi 6.70, W did not
changed remarkably. In C. japonicus, L increased by ca
10% but W scarcely change at pHo 6.8.
When the fertilized eggs were treated after NEBD
with normal pHSW (pHo 6.2–8.0) which contained
neither acetate nor ammonia, they divided normally and
no morphological change in their mitotic apparatus was
observed during division. Because the treatment with
normal pHSW at pHo 6.2–8.0 did not induce any pHi
change as described in the previous subsection, the
obtained results are quite reasonable.
Birefringence retardation of the mitotic spindle.
As shown in Figure 4 and Table III, the retardation of the
spindle was measured by polarization microscopy in the
eggs treated with Ac-pHSW at pHo 6.8 and 7.0. The
retardation increased both at pHo 6.8 and 7.0 (pHi 6.70
and 6.77, respectively) compared to that at pHi 7.3 of
control eggs. According to Hiramoto et al. [1981b],
microtubule number in cross-section of the spindle was in
S. mirabilis calculated to increase by ca 20% at pHo 6.8
than that at pHi 7.3, which is in good agreement with the
increase in width as mentioned above. Consequently,
total length of microtubules in the metaphase spindle in S.
mirabilis increased by ca 40% with regard to increasing
by 20% in the length, assuming that the spindle is an
ellipsoid of revolution whose revolving axis is a pole to
pole line.
Watanabe et al.
Persistent Metaphase in the Treated Eggs
at Low pH
After treating the eggs with Ac-pHSW at pHo 6.8
and 7.0, some eggs did not divide and the division
appeared to retard, whereas the eggs treated after NEBD
with NH3-pHSW and NH31Ac-pHSW at the higher pH
did not show any difference from the control eggs. In
order to examine the cause of division retardation, the
eggs were lysed and the mitotic stage of each egg was
determined, and then the rate of the eggs at prophase or
prometaphase, metaphase, and anaphase or telophase was
measured with time after the treatment with Ac-pHSW at
pH 6.8 and 7.0. As shown in Figure 5a and b, the
population at prophase or prometaphase of the treated
eggs was decreased in a similar manner to that of control.
However, the eggs at metaphase with treatment increased
in number with time, and on the contrary, the eggs at
anaphase or telophase did not increase, which indicates
that metaphase was persistent in the treated eggs. Some of
these acidified eggs divided within 30 min of the treatment, and the other did not divide within 1 hour or more
of the treatment, suggesting that only the eggs which did
not stop at metaphase could divide at acidic pH. On the
other hand, alkalized eggs usually divided, although the
division was somewhat delayed at high pH.
In this study, when the fertilized eggs were treated
with acidified sea water containing acetate and/or ammonia (Ac-pHSW, NH3-pHSW, and NH31Ac-pHSW) at
various pH values, pHi shifted to acidic or alkaline pH,
which resulted in the change in the size of the mitotic
spindle. This change may be attributed to the total length
of microtubules in the spindle detected by immunofluorescence and polarization microscopy. Therefore, it is suggested that slightly acidic pH favored microtubule assembly and, on the contrary, alkaline pH favored microtubule
disassembly. These results are explained as follows. It is
known that the stability of microtubules and critical
concentration for microtubule assembly are maximal at
slightly acidic pH using purified brain and egg tubulin
[Olmsted and Borisy, 1975; Regula et al., 1981; Suprenant, 1989; Simons et al., 1992; Tiwari and Suprenant,
1994]. Moreover, Deery and Brinkley [1983] reported
that at a slightly acidified condition, microtubules assembled from endogeneous tubulin in the cell which was
lysed and pretreated with colcemid. However, slightly
alkaline pH, but not acidic pH, favored microtubule
assembly in the crude extracts of eggs or oocytes
[Suprenant and Marsh, 1987; Suprenant, 1989, 1991;
Simons et al., 1992; Tiwari and Suprenant, 1994]. Tiwari
and Suprenant [1994] explained this phenomenon by the
hypothesis that there would be some pH-sensitive protein
Fig. 5. Persistent metaphase of the eggs treated with Ac-pHSW at pHo
6.8. a: Rates of the eggs at prophase or prometaphase, metaphase, and
anaphase or telophase to total eggs are shown in the ordinate and the
abscissa is time after treatment in minute when the eggs were treated
with Ac-pHSW at pHo 6.8. b: Rates of the eggs at prophase or
prometaphase, metaphase, and anaphase or telophase to total eggs are
shown in the ordinate and the abscissa is the same time after treatment
in minute as that in a. Pro1prometa and ana1telo indicate prophase or
prometaphase and anaphase or telophase, respectively. The rates of
treated eggs and those of control eggs are shown in closed and open
symbols, respectively. The rates of prophase or prometaphase, metaphase, and anaphase or telophase are totally 100% at every time after
the treatment. Sample number of each point is 65 to 93.
which binds to tubulin at acidic pH in the crude extracts,
decreases the amount of tubulin to polymerize and then
lowers the amount of microtubules. According to this
hypothesis, in the living fertilized egg, the pH-sensitive
protein may be inactivated by some mechanism and,
therefore, the property of microtubule assembly in the
Effects of pHi on Mitosis in Sand Dollar Egg
living egg is similar to that in purified tubulin solution.
There are some other explanations to induce the spindle
elongation or shortening. The pH changes alters the
activity of some microtubule-based motors because spindle
organization is induced by some motor proteins [Veros
and Karsenti, 1996]. The pH changes may also affect the
nucleation capacities of the centrosome or the stabilizing
activity of the kinetochore.
At pHi of 6.3, the mitotic apparatus was poorly
organized. This result is agreeable with the reports that
the amounts of assembled microtubules were small at
more acidic pH than pH 6.6–6.8 [Olmsted and Borisy,
1975] and that at low pH of 5.8 to 6.2, ribbon- or
sheet-like polymers were polymerized from tubulin instead of microtubules [Matsumura and Hayashi, 1976].
When the mitotic apparatus was not organized, the cell
did not divide. Schatten et al. [1985] reported that
microtubules were unable to assemble shortly after
fertilization in acidified sea urchin eggs and disassembled
even at pHi of 7.0. Their results were not in agreement
with ours in the present study. At this time, the reason
why our results differed from theirs is unknown.
In this study, slightly acidic pHi induced persistent
metaphase, which resulted in cleavage inhibition. In this
condition, the mitotic apparatus was not disorganized but
increased in size, which indicates that the mitotic microtubules were stabilized at the pHi. It is known that in the
cell treated with microtubule stabilizing reagents, hexylene glycol and taxol, anaphase chromosome movement
was not induced and cleavage was inhibited [Hamaguchi
et al., 1987; Oka and Hamaguchi, 1991]. It is possible that
stabilization of microtubule directly inhibits anaphase
chromosome movement. However, we can not exclude
the possibility that pH changes reduced some factors
involving metabolic activities which would progress the
mitotic stage from metaphase to anaphase.
We express our gratitude to the staff of the Ushimado Marine Biological Station of Okayama University
and to the staff of the Misaki Marine Biological Station of
the University of Tokyo for supplying sand dollars.
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acid, inhibits, ethacrynic, ethylmaleimide, kinesin, motility, binding, microtubules, assays
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