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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 INTRODUCTION 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 264 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. MATERIALS AND METHODS 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) Composition Normal pHSW Ac-pHSW NH3-pHSW Ac1NH3-pHSW CH3COONa NH4Cl NaCl KCl MgCl2 MgSO4 EGTA PIPES HEPES pH range 0 0 436 9 34 16 1 5 5 6.2–8.0 10, 20 0 426, 416 =a = = = = = 6.2–7.3 0 10, 20 426, 416 = = = = = = 7.3–8.0 20 20 396 = = = = = = 7.3 aArrow shows the same figure as the left column. pH was adjusted by NaOH. 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  and Hamaguchi . 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 calculated. 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. Immunofluorescence 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 265 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 pHSW pHo n pHi before treatment AC-pHSW 6.20 6.50 6.80 7.00 7.30 7.30 7.30 7.70 8.00 1 3 11 4 16 4 15 5 4 7.37 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 AC1NH3-pHSW NH3-pHSW pHi after treatment (10 min) 6.30 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. 266 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 267 TABLE III. Birefringence of the Mitotic Apparatus Treated With Ac-pHSW Widtha (µm) Control Ac-pHSW (pHo 6.8) Ac-pHSW (pHo 7.3) Scaphechinus mirabilis Ra MNa (nm) (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 4.8 5.7 5.0 (n) Width (µm) (13) (17) (20) 9.0 6 0.7 9.0 6 1.1 — Clypeaster japonicus R MN (nm) (3103 ) 4.4 6 0.2 5.0 6 0.5 — 4.5 5.0 — (n) (12) (10) aWidth is spindle width; R is birefringence retardation (nm); and MN is number of microtubules in the cross-section of the mitotic spindle. RESULTS 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. 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. 268 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. DISCUSSION 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  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  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.  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. 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