Proc. Natl. Acad. Sci. USA Vol. 91, pp. 3921-3925, April 1994 Cell Biology Metaphase and anaphase in the artificially induced monopolar spindle (mitosis/chromosome movement/mitotic spindle/sea urchin egg) KoHnI ITO*, MICHITAKA MASUDAt, KEIGI FUJIWARAt, HIROSHI HAYASHI*, AND HIDEMI SATOf *Sugashima Marine Biological Laboratory, School of Science, Nagoya University, Toba, Mie 517, Japan; tDepartment of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan; and *School of Science, Nagoya University, Nagoya 464, Japan Communicated by Daniel Mazia, January 3, 1994

ABSTRACT By using monopolar spindles artificially in- and a period clearly defined as metaphase was not observed duced in sea urchin embryos, we exmied whether or not the (5, 6, 11). Leslie (12) produced an extra half-spindle exper- presence of two opposing poles was an indispensable condition imentally from one pole of a bipolar spindle in fertilized sea for keeping chromosomes at a fixed distance from the pole at urchin eggs. Statistical analyses using fixed cells showed that metaphase and for the anaphase chromosome movement. the average chromosome-to-pole distance in the metaphase Chromosomes were stained with Hoechst dye 33342 and their extra half-spindle was identical to that in the metaphase behavior was followed in the monopolar and the control bipolar bipolar spindle. Monopolar spindles were also produced in a spindles. In the monopolar spindle, chromosomes were first temperature-sensitive mutant line of Syrian hamster cells arranged on a curved metaphase plate and then spread on a (13). Electron microscope studies of these cells showed that part of the Imaginary surface of a sphere whose center was the chromosomes lined up in a metaphase-like position in the monopole. The estimated chromosome-to-pole distance was monopolar spindle. However, since chromosome movement similar to that of bipolar spindies at metaphase and remained was not followed in living cells in either of these studies, an fixed until chromosomes started to move toward the pole. The oscillatory chromosome movement similar to that in newt average duration of metaphase in the monopolar spindle was 6 lung cells could not be ruled out. times longer than that in the bipolar spindle. The poleward Monopolar spindles can be artificially induced in sea urchin movement of chromosomes in the monopolar spindle was embryo cells (14) and have been extensively characterized: similar to the anaphase A (chromosome-to-pole movement) in one cell has one centrosome consisting of a pair ofcentrioles the bipolar spindle with respect to the velocity, duration, (15); the single pole organizes chromosomes in a quasi- distance, and synchronization ofmigration. These results show metaphase arrangement in which one kinetochore facing the that even half of the normal spindle has capacities for the pole is connected to the pole by a bundle of microtubules, arrangement of chromosomes at metaphase and for the whereas its sister kinetochore facing away from the pole is anaphase A chromosome movement. Based on these results, we free from microtubule contact (16). In spite of these detailed were able to exclude some existing theories of metaphase, such morphological data, chromosome movement in the mono- as the one based on the balance offorces between the two poles. polar spindle has not been convincingly described, because it is difficult to identify chromosomes in monopolar spindles in There are several hypotheses on the mechanism for the living sea urchin blastomeres with a Nomarski or a phase- arrangement of chromosomes on the metaphase plate. Per- contrast microscope. The same problem also makes it diffi- haps, the most conventional view (hypothesis A) states that cult to determine whether or not a stable metaphase exists in the balance between two poleward forces at the oppositely the monopolar spindle. oriented kinetochores keeps paired sister chromatids at the To overcome these difficulties, we stained chromosomes midpoint between two poles (1-3). Another hypothesis (hy- in artificially induced sea urchin monopolar spindles with pothesis B) is that the chromosomes are confined to the Hoechst dye 33342 and observed chromosome behavior in equatorial boundary of a spindle because this is the equilib- living cells. Our analysis has shown that a period definable as rium position between the poleward force at the kinetochore metaphase exists, indicating that the balance offorces due to and the resistance of the relatively solid half-spindle to the the presence oftwo opposite poles as proposed by hypothesis further advance of the chromosomes to the pole (4). Instead A is not indispensable for the establishment of metaphase. of a physical boundary, hypothesis C postulates that each Our study has suggested that the astral ejection force acting chromosome arm is subjected to an ejection force generated on chromosome arms as proposed by hypothesis C is not a by astral microtubules to balance the poleward force at the significant force at metaphase. As the mechanism for estab- kinetochore (5-7). A newer idea (hypothesis D) is that lishing the metaphase chromosome arrangement, our results chromosomes are kept at the metaphase plate by the regu- are compatible with hypotheses B and D. In addition, lation of microtubule-based motors located at the kineto- anaphase chromosome movement, which was indistinguish- chore (8-10). able from anaphase A in bipolar spindles, was observed in the A monopolar spindle is an interesting in vivo system for monopolar spindle, indicating that the monopolar spindle investigating how chromosomes become arranged at a fixed also possessed the complete machinery required for the distance from the pole during metaphase. Chromosomes in a anaphase A chromosome movement. monopolar spindle cannot attain a metaphase position by the balance oftwo opposing forces as proposed by hypothesis A, but they may do so by the mechanism proposed by hypoth- MATERIALS AND METHODS esis B, C or D. In monopolar spindles of cultured newt lung Induction ofMonopolar Spindles. Monopolar spindles were cells, chromosomes oscillate toward and away from the pole induced in sea urchin eggs as described by Mazia et al. (14). In brief, fertilized eggs of the sea urchin (Hemicentrotus The publication costs ofthis article were defrayed in part by page charge pulcherrimus) and the sand dollar (Clypeaster japonicus) payment. This article must therefore be hereby marked "advertisement" were immersed in artificial seawater (Jamarin U, Osaka) in accordance with 18 U.S.C. §1734 solely to indicate this fact. containing 0.1 M 2-mercaptoethanol at prometaphase-to- 3921 Downloaded by guest on September 30, 2021 3922 Cell Biology: Ito et al. Proc. Natl. Acad. Sci. USA 91 (1994) metaphase of the first division and were kept immersed until Immunofluorescence Microscopy. Blastomeres having the control eggs completed the second division. They were monopolar spindles were fixed by incubation with 0.1% then washed several times with artificial seawater to remove glutaraldehyde and 2% formaldehyde in microtubule- 2-mercaptoethanol. Each egg formed a tetrapolar spindle and stabilizing medium [10 mM EGTA/0.55 mM MgCl2/25% divided into four daughter cells (1:4 division). Each daughter (wt/vol) glycerol/1% (wt/vol) Nonidet P-40/0.5 mM phenyl- cell reformed the nucleus and went into the next cell cycle, methanesulfonyl fluoride/25 mM Mes; pHl 6.8 (18)] for 20 during which a monopolar spindle was formed. In some cells, min. Microtubules were indirectly immunostained with rab- one of the furrows failed, so that they divided into three bit anti-tubulin antiserum raised against vinblastine-induced daughter cells (1:3 division). In this case, one cell had a sea urchin tubulin crystals (19) and followed by fluorescein- bipolar spindle and the other two cells had monopolar spin- conjugated affimity-purified goat anti-rabbit IgG (Cappel). dles. The induction ofmonopolar spindles was ascertained by Chromosomes were counterstained with propidium iodide at using a supersensitive polarizing microscope (17) manufac- 0.1 pg/ml. An Olympus confocal laser scanning microscope tured by Nikon Engineering. (LSM-GB200) was used to obtain optical sections of the Observation of Chromosome Movement. After 2-mercapto- monopolar spindle. Three-dimensional images were con- ethanol treatment, cells were immersed in artificial seawater structed by using a computer program developed by S. Hanai with Hoechst 33342 at 5-10 pg/ml. Embryos stained with the (Department of Vascular Physiology, National Cardiovascu- fluorescent dye developed normally at least to the hatching lar Center Research Institute, Osaka). stage. Chromosome movement was observed by using an Olympus BH-2 epifluorescence microscope equipped with a RESULTS 100-W mercury lamp, a standard UV filter set (excitation, 365-nm band pass; emission, 435-nm long pass) and a x40 Prometaphase in Monopolar Spindles. We observed chro- oil-immersion objective lens (na, 1.0). A neutral density filter mosome movement in both monopolar and control, bipolar (ND8) and a heat-cut filter were used to minimize photo- spindles in living sea urchin embryo cells which were stained bleaching and cell damage. Fluorescence images were col- with Hoechst 33342. Immediately after the breakdown ofthe lected by a SIT camera (C-1000-12, Hamamatsu Photonics, nuclear envelope, fluorescent chromosomes were seen to be frames an scattered inside the spherical region where the nucleus had Hamamatsu, Japan), averaged over four to eight by occupied. In the bipolar spindle of H. pulcherrimus, at 18 ± image processor (Avio Image, Nippon Avionics, Tokyo), and 1C, chromosomes became arranged on the metaphase plate stored on video tape. The total time ofobservation ofone cell about 10 min after nuclear envelope breakdown. They re- was limited to about 7 min because illuminating light caused mained stationary on the metaphase plate for 3-4 min, and no detectable effect on chromosome movement in control then anaphase chromosome movement ensued. In the mono- bipolar spindles at least within this time span. Photographs of polar spindle, chromosomes did not align in a plane, defined chromosomes were taken directly from the video monitor on as the metaphase plate in the bipolar spindle. Instead, they Kodak Panatomic X film or T-MAX 100 film. were arranged on a curved metaphase plate (Fig. 1) as Mazia Data Analysis of Chromosome Movement. To follow the et al. (4, 16) reported earlier. The time from nuclear envelope movement ofchromosomes in a monopolar spindle, the x and breakdown to this arrangement of chromosomes was about y coordinates ofeach kinetochore and the pole were digitized 10 min, which was identical to the duration ofprometaphase with an image analyzer (L525, Pias, Osaka), and the kineto- in the bipolar spindle. chore position of each chromosome relative to the pole was Stable Chromosome-to-Pole Distance in Metaphase Mono- determined. The kinetochore was defined as the portion of a polar Spindles. Once chromosomes were arranged on a chromosome facing the pole during anaphase (see Fig. 4). curved metaphase plate in the monopolar spindle, they did The position of the pole was determined from fluorescence not oscillate toward and away from the pole, although they images as the center of a spherical, less fluorescent area (see could move laterally. As a result, chromosomes became Fig. 2a) which was formed by the exclusion of Hoechst distributed on a part (more than half) ofthe imaginary surface dye-stainable cytoplasmic granules from the region occupied of a sphere whose center was the pole (Fig. 2a). The by the monopolar spindle. This position corresponded well chromosome-to-pole distance remained constant for several with the pole determined by polarization microscope images minutes to over an hour (Fig. 3 and see below) until the onset (see Fig. 2b). Spindles in cells at the second and the third of anaphase-like chromosome movement. For H. pulcherri- mitosis were used as control bipolar spindles because the cell mus, this distance was 9.7 ± 0.9 jim (30 cells, 18 ± 1PC), volume of these cells was comparable to that of cells with a which was similar to the chromosome-to-pole distance in the monopolar spindle. metaphase bipolar spindle ofthe third mitosis (9.8 ± 0.3 pm;

FiG. 1. Stereo pairofmicrographs ofa monopolar spindle at early metaphase. A cell having amonopolar spindle was fixed, and chromosomes were stained with propidium iodide. Microtubules were stained with rabbit anti-tubulin followed by fluorescein-labeled anti-rabbit IgG. Three-dimensional images were reconstructed from images obtained by confocal laser microscopy. The bright fluorescent spots outside the spindle area are particulate structures on the cell surface that were stained nonspecifically by both fluorescent reagents. (Bar = 10 pm.) Downloaded by guest on September 30, 2021 Cell Biology: Ito et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3923 data indicated that the time from nuclear envelope break- down to telophase in the monopolar spindle was on the average about 3 times longer and was much more variable than that in the bipolar spindle (monopolar spindles, 35.5 ± 20.9 min, n = 33; bipolar spindles, 12.7 ± 1.6 min, n = 39; C. japonicus at 22 ± PC). Similar results were reported by Sluder and Begg (20). As earlier suggested by Sato (21), this prolonged and varied progression time in monopolar spindles seemed to be entirely due to variable duration of metaphase, since we found that the durations of both prometaphase and anaphase in the monopolar spindle were the same as those of the bipolar FIG. 2. (a) Fluorescence microscope image of a living sea urchin embryo cell having a monopolar spindle at middle/late metaphase. spindle (prometaphase, see above; anaphase, shown below). Chromosomes were stained with Hoechst 33342, and the focal plane The calculated average duration of metaphase in the mono- is at the level of the pole. Chromosomes are arranged in part of a polar spindle was 27.0 ± 20.9 min (n = 33, coefficient of circle. The center of the circular, less fluorescent area in the cell variation = 0.77, C. japonicus) and was 6 times longer and 3 corresponds to the position of the pole determined by polarization times more variable thanthatfor the bipolar spindle (4.3 ± 1.2 microscope in b. (b) Polarization microscope image of the same cell min, n = 33, coefficient of variation = 0.28). Occasionally, shown in a. (Bar = 20 pm.) two or more monopolar spindles were formed in a cell. Iftwo monopolar spindles were close together, they fused and made 46 cells) although the standard deviation for the monopolar a bipolar spindle and immediately went into anaphase, as spindle was larger than that for the bipolar spindle. The larger H.S. and D. Mazia (unpublished observation) and Sluder and deviation could be partly explained by nonuniform blas- Begg (20) earlier observed. These results imply that bipolarity tomere size produced by 1:4 or 1:3 division. These results of the spindle favors a smooth transition from metaphase to show that a period during which chromosomes are at a fixed anaphase. distance from the pole exists in the monopolar spindle. This Poleward Chromosome Movement in Monopolar Spindles. period may be analogous to metaphase. Thus, our results After prolonged metaphase, all chromosomes in a monopolar suggest that the mechanisms for determining and maintaining spindle started to move toward the pole in unison (Fig. 4). At the constant chromosome-to-pole distance during metaphase the same time, some chromosomes started to rotate (Fig. 4, can function with only one pole. arrow) or bend (arrowhead). During chromosome movement, Prolonged Metaphase in Monopolar Spindles. In bipolar the portion of the chromosome which oriented toward the spindles, a constant duration of metaphase was observed. pole by the above-mentioned rotation or bending kept its For example, in the second division of C. japonicus eggs, leading position. This rotation or bending most likely repre- metaphase lasted 4.3 ± 1.2 min at 22 ± 1C (39 cells). sents the motion involved in repositioning ofthe kinetochore Following the behavior of the fluorescently labeled chromo- toward the pole. Chromosomes did not go all the way to the somes in monopolar spindles, we found that the duration of pole but stopped when they reached =6 pam from the pole. At metaphase was much longer and more variable compared that time, the birefringence of the monopolar spindle dimin- with bipolar spindles. However, we could not measure the ished to the telophase level ofcontrol, bipolar spindles. Soon exact duration of metaphase by this method, because cells after the end of chromosome movement, the nuclear mem- could not withstand a long-term exposure to the excitation brane was re-formed. light of an epifluorescence microscope. Therefore, we deter- We analyzed the velocity, duration, and distance of the mined the duration of metaphase by subtracting the duration poleward chromosome movement (Fig. 5). These three pa- of both prometaphase and anaphase (described below) from rameters in the monopolar spindle were similar to those of the total time between nuclear envelope breakdown and anaphase A chromosome movement in the bipolar spindle telophase. The time of nuclear envelope breakdown in a cell (Table 1). These results suggest that the poleward chromo- was determined by using a Nomarski differential interference some movement in monopolar spindles is anaphase A chro- contrast microscope. The beginning of telophase in the same mosome movement and further suggest that a half-portion of cell was detected by a polarizing microscope as an abrupt the mitotic apparatus possesses all the necessary elements for decrease in the birefringence of the monopolar spindle. Our anaphase A chromosome movement. A notable difference was that unlike anaphase A in bipolar spindles, the segrega- - 12 tion of sister chromatids was not observed in monopolar 11 spindles. Lack of chromosome segregation in the sea urchin 10 -j-_-_~~~~~~~~~~~~~~~~~~~~0ZZ . monopolar spindle was reported by Mazia et al. (16). They N also reported splitting apart of chromosomes, but the reso- 0 lution of our fluorescence image of the chromosome was o. 8 insufficient to confirm this. E7 0 DISCUSSION (a0 6 E In this study, we have examined chromosomal movement in 2 5 monopolar spindles in living sea urchin embryos and dem- C. A onstrated that stages clearly definable as metaphase and 0 50 100 150 200 250 300 350 anaphase exist in the absence of an opposing spindle pole. Time (sec) Chromosomes were first arranged on a curved metaphase plate and then three-dimensionally arranged around the FIG. 3. Chromosome-to-pole distance ii a monopolar spindle during metaphase. The distance (determinedI as described in Mate- monopole. The chromosome-to-pole distance was the same rials and Methods) is constant during metap]hase. The graph shows as that of the control, bipolar spindle at metaphase and that the position of four chromosomes (different symbols) does not remained constant until the onset of anaphase, during which change relative to the pole. Time 0 indicateis the beginning of the they moved in the same manner as in anaphase A of the fluorescence microscope observation. bipolar spindle. Our results provide direct evidence that a Downloaded by guest on September 30, 2021 3924 Cell Biology: Ito et aL Proc. Nati. Acad. Sci. USA 91 (1994)

14 13 c 0000 0 12 09 a. 11 o92 10 - 10 9 8 \ oooo 00°°oo0o E 0 at C) A _- -50 0 50 100 150 200 250 300 Time (sec)

Fio. 5. Time course of anaphase chromosome movement in the monopolar spindle. Two chromosomes located in the same focal plane as the pole were followed. These chromosomes are indicated i in Fig. 4 by the arrow (o) and arrowhead (e). The relative position AI ofeach chromosome to the pole was determined by use of an image analyzer. The velocity of chromosome movement was calculated from the linear portion of the graph, lasting about 60 sac, by linear regression. The chromosomes moved at 2.5 in/min (o) and 2.7 psm/min (e). Anaphase lasted 170 sec (o) and 140 sec (e).

shape) as if they are being pushed away from the pole (5, 6, 11). To explain these observations, Rieder and coworkers (5, 6) proposed hypothesis C, suggesting that a poleward force acting at the kinetochore was balanced with an antipoleward force acting on chromosome arms. The antipoleward force was assumed to be generated by growing astral microtubules and was termed the astral ejection force (5, 6). The same mechanism was also suggested to explain the metaphase-like positioning of chromosomes in an extra half-spindle of fer- tilized sea urchin eggs (12). Our results do not agree with hypothesis C, however. Ifthe balance of forces proposed in this hypothesis were the FiG. 4. Anaphase chromosome movement in a monopolar spin- primary mechanism of metaphase in monopolar spindles, an dle, shown by sequential fluorescence images of the same cell. Time anaphase-like chromosome configuration, such as the V is expressed in seconds (-75 to 170) with time 0 representing the shape for a metacentric chromosome, would be expected. A onset of chromosome movement. At the beginning of chromosome telocentric chromosome [most sea urchin chromosomes are some chromosomes start to rotate or bend movement, (arrow) telocentric (22)] would position itself so that its kinetochore (arrowhead). This rotation or bending most likely represents repo- sitioning of the kinetochore toward the pole. The putative kineto- end is closest to the pole and the other end away from the chore positions of two chromosomes are shown by an arrow and an pole. However, we observed that metacentric chromosomes arrowhead. During anaphase chromosome movement, the kineto- were nearly straight at metaphase, although they did become chore orientation of each chromosome does not appear to change, V-shaped in anaphase (Fig. 4, arrowhead). Furthermore, although the bending of the metacentric chromosome becomes some chromosomes, presumably telocentric ones, quickly sharper and sharper. (Bar = 10 pm.) rotated at the onset ofanaphase, indicating that they were not aligned as expected during metaphase (Fig. 4, arrow). Thus, single pole is sufficient to achieve basic chromosome orga- chromosomes are not necessarily oriented as hypothesis C nization and motion in mitosis. However, in the monopolar predicts. Our results suggest that in sea urchin embryo cells, spindle, the smooth transition from metaphase to anaphase the astral ejection force is not a significant force in meta- and the segregation of sister chromatids were not observed, phase. However, this conclusion is based on the assumption suggesting that these events are somehow dependent on the that the rheological properties both of chromosomes and of presence of two opposing poles. the surrounding cytoplasm do not change significantly during Our present results clearly oppose hypothesis A (see metaphase to anaphase. Introduction), which states that a balance between antago- Our results are not incompatible with hypotheses B and D. nistic poleward forces at opposing kinetochores keeps chro- Hypothesis B, proposed by Mazia (4), states that metaphase mosomes at the metaphase position (1-3). According to this chromosomes are held on the surface of the monopolar hypothesis, metaphase should not exist in the monopolar spindle because a monopolar spindle is (i) an isolatable spindle, but our study has proved otherwise. In fact, Mazia coherent structure, (ii) densely packed with membranes and (4) pointed out in his recent review that metaphase in the vesicles, and (iii) not merely a space through which micro- monopolar spindle could not be explained by the balance of tubules run (16). Thus, it is possible to imagine that chromo- two opposing forces. How can metaphase be achieved in the somes cannot penetrate into this relatively solid structure. monopolar spindle? In monopolar spindles in cultured newt Hyman and Mitchison (8) have shown the presence of two lung cells, chromosomes are not locked into a stable position microtubule-based motors, one for a poleward and the other but oscillate toward and away from the single pole (5, 6, 11). for an antipoleward chromosome movement, at the kineto- In spite of the oscillatory motion, the average chromosome- chore and suggested that a kinase-phosphatase system is to-pole distance is similar to that in normal bipolar spindles involved in the control of the direction of chromosome at metaphase (11). In addition, chromosome arms in the movement. Their proposed model (hypothesis D) for meta- monopolar spindle bend radially away from the pole (V phase chromosome alignment states that a kinetochore de- Downloaded by guest on September 30, 2021 Cell Biology: Ito et al. Proc. Natil. Acad. Sci. USA 91 (1994) 3925 Table 1. Quantitative aspects of anaphase A Chromosome-to-pole Distance of distance, AM migration, Duration, Velocity, Cell n Metaphase Telophase Am sec jptm/min Monopolar spindle 1 6 9.3 6.1 3.2 150 1.1 2 3 9.2 5.2 4.0 150 1.6 3 4 10.2 7.2 3.0 130 1.6 4 3 10.1 6.2 3.9 210 1.2 5 2 12.5 7.9 4.6 160 2.6 Average 10.3 6.5 3.8 160 1.6 Bipolar spindle (second mitosis) 1 6 11.5 8.3 3.2 120 2.0 2 6 11.7 6.7 5.0 190 1.7 3 3 10.6 6.4 4.2 180 1.8 4 2 12.1 9.1 3.0 170 1.2 5 3 11.3 7.1 4.2 160 1.5 6 6 11.0 6.4 4.6 170 1.5 7 2 10.8 4.3 4.3 140 1.9 Average 11.3 6.9 4.4 160 1.7 Embryos ofH. pulcherrimus were kept at 18 + 1PC. Movement ofchromosomes located in the same focal plane as the pole and identifiable individually was continuously followed by fluorescence microscopy. For each cell, an average value calculated from n (no. of chromosomes) observations is given for each parameter. termines chromosome position by regulating the activity of that the basic autonomous unit in mitosis is a monopolar the motors according to a certain gradient of information spindle and that a bipolar spindle is composed of two mono- from the poles (8, 9). This model can provide an explanation polar spindles (4). We believe that the monopolar spindle for the presence of stable metaphase and anaphase A chro- induced in sea urchin embryos is a valuable model for mosome movement in monopolar spindles. studying the mechanism of mitosis. As discussed above, our results are contradictory to two popular hypotheses for the mechanism of metaphase, hy- We gratefully acknowledge Professor Daniel Mazia for inspiring potheses A and C. While observations made on the sea urchin two of us 20 years ago and for not only having encouraged our research but also giving us valuable and imaginative advice. This monopolar spindle appear to leave little room for any hy- work was supported in part by Grants-in-Aid for Scientific Research potheses based on the balance of forces coming from two (01540593 and 62890004) from the Ministry of Education, Science, poles, it is still possible that different organisms rely on and Culture, Japan. different mechanisms for metaphase chromosome alignment. It is also possible that cells use multiple regulatory and 1. McNeil, P. A. & Berns, M. W. (1981) J. CellBiol. 88, 543-553. force-generating mechanisms for mitosis (23) and that this 2. Hays, T. S. & Salmon, E. D. (1990) J. Cell Biol. 110,391-404. redundancy creates discrepancies among different experi- 3. Ostergren, G. (1951) Hereditas 37, 85-156. mental systems because each experimental system or orga- 4. Mazia, D. (1987) Int. Rev. Cytol. 100, 49-92. nism emphasizes one mechanism over others. For example, 5. Rieder, C. L., Davison, E. A., Jensen, L. C. W., Cassimeris, the bipolar spindle may employ auxiliary mechanisms based L. & Salmon, E. D. (1986) J. Cell Biol. 103, 581-591. 6. Ault, J. G., Demarco, A. J., Salmon, E. D. & Rieder, C. L. on the balance oftwo counterforces from two opposing poles (1991) J. Cell Sci. 99, 701-710. in addition to the one which works only with a single pole. In 7. Salmon, E. D. (1989) in Cell Movement, ed. Warner, F. D. & cultured newt lung cells, chromosomes are much larger than McIntosh, J. R. (Liss, New York), Vol. 2, pp. 431-440. those of sea urchin embryos, and accordingly the ejection 8. Hyman, A. A. & Mitchison, T. J. (1991) Nature (London) 351, force experienced by newt chromosomes may also be signif- 206-211. icant. As a result, chromosome configuration in monopolar 9. Mitchison, T. J. (1989) in Cell Movement, ed. Warner, F. D. & spindles in these cells may be V-shaped during metaphase. McIntosh, J. R. (Liss, New York), Vol. 2, pp. 421-430. Our data suggest that there is a mechanism working at 10. Skibbens, R. V., Skeen, V. P. & Salmon, E. D. (1993) J. Cell metaphase either for determining the length of kinetochore Biol. 122, 859-875. 11. Bajer, A. S. (1982) J. Cell Biol. 93, 33-48. microtubules or for locating kinetochores at a fixed distance 12. Leslie, R. J. (1992) J. Cell Sci. 103, 125-130. from the pole in the monopolar spindle in sea urchin embryo 13. Wang, R. J., Wissinger, W., King, t. J. & Wang, G. (1983) J. cells. Further, our study suggests that by such a mechanism, Cell Biol. 96, 301-306. the metaphase chromosome arrangement can be accom- 14. Mazia, D., Harris, P. J. & Bibring, T. (1960) J. Biophys. plished independent ofany interaction between two opposing Biochem. Cytol. 7, 1-20. poles or the balance of forces acting on different portions of 15. Sluder, G. & Rieder, C. L. (1985) J. Cell Biol. 100, 887-896. a chromosome. Our results demonstrate that monopolar 16. Mazia, D., Paweletz, N., Sluder, G. & Finze, E.-M. (1981) spindles can go through anaphase with the same kinetic Proc. NatI. Acad. Sci. USA 78, 377-381. property as anaphase A in bipolar spindles. This fact indi- 17. Sato, H., Takenaka, H. & Niboshi, T. (1991) Acta Histochem. Cytochem. 24, 343-347. cates that any interaction between antiparallel microtubules 18. Balczon, R. & Schatten, G. (1983) Cell Motil. 3, 213-226. emanating from two opposing poles is not necessary either 19. Fujiwara, K. & Pollard, T. D. (1978) J. Cell Biol. 77, 182-195. for the force generation or for the velocity control of 20. Sluder, G. & Begg, D. A. (1983) J. Cell Biol. 97, 877-886. anaphase A chromosome movement. It also indicates that the 21. Sato, H. (1982) Cell Differ. 11, 345-348. velocity of chromosome movement is controlled within each 22. Saotome, K. (1987) Zool. Sci. 4, 483-487. half of a bipolar spindle. Our observation supports the idea 23. Goldstein, L. S. B. (1993) J. Cell Biol. 120, 1-3. Downloaded by guest on September 30, 2021