Printed in Sweden Copyright (9 1975 by Academic Press, Inc. All rights of reproduction in any form reserued
Experimental Cell Research91 (1975) 57-62
KINETOCHORES
AND AND
MICROTUBULES CHROMOSOME
IN MULTIPOLAR
MITOSIS
ORIENTATION
W. K. HENEEN Institute of Genetics and The Wallenberg Laboratory, University of Lund, S-223 62 Lund, Sweden
SUMMARY The ultrastructure of the kinetochore and the orientation of kinetochore microtubules were studied in multipolar divisions of cultured rat-kangaroo cells (Pt-Kl). The metaphase kinetochore exhibited a lamellar structure and most of the chromosomes expressed a bipolar microtubule orientation. A multipolar kinetochore orientation was observed in some chromosomes. Equal or unequal portions of a chromatid’s kinetochore and a corresponding number of kinetochore microtubules may be oriented to two different poles. Curved or bent continuous microtubules were observed in the vicinity of chromosomes showing multipolar orientation. The findings are in accordance with bstergren’s theory of ‘auto-orientation’ [S]. It is speculated that orientation of a chromatid kinetochore to more than one pole might be a possible regular event during the process of chromosome orientation prior to full metaphase in bipolar mitosis.
The kinetochore organization as well as the dynamics of chromosome movement and orientation are major topics of interest when dealing with processes of mitosis and meiosis. The present knowledge in this area is based mainly on light microscopic, cinematographic and, more recently, electron microscopic observations [l-4]. Ultrastructural analysis elucidates important aspects such as the detailed appearance of kinetochores, kinetochore/microtubule interrelationships and directions of individual microtubules. Elucidation of these features could be instructive for an understanding of chromosome movement and orientation. The metaphase kinetochore in mammalian materials is characterized by its lamellar appearance which was interpreted to represent a disc [5, 61 or a filamentous structure [l, 4, 71. Regular bipolar divisions were used in the majority of the studies that
deal with the fine structure of the mitotic apparatus. In general, each of the two sister metaphase kinetochores has microtubule connections with only one pole. ‘Wrong’ or ‘forbidden’ connections between one sister kinetochore and two polar regions or between two sister kinetochores and one polar region have also been documented both in mitosis and meiosis [8-lo]. The aim of the present investigation was to find out about the appearance and orientation of kinetochores in multipolar mitosis. MATERIALS
AND METHODS
The rat-kangaroo (Potorous tridactvlisj cell line Pt-Kl was used. Multipolar divisions are ’ frequently encountered (13.1 X) in this cell line which is also characterized by its flat mitotic cells [II]. The stemline chromosome number is 11 ($!,2n = 12). Cells were grown in Falcon plastic flasks and fixed and embedded using the technique of Brinkley et al. [12]. Cells in desirable mitotic stages were cut using an LKB microtome. The sections were stained with Exptl Cell Res 91 (1975)
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Fig. I. Multipolar metaphases in cultured rat-kangaroo (Potorous triductylis) cells (Pt-Kl) shaped, or (b) T-shaped equatorial plates. Solid and dashed lines indicate directions dashed lines represent possible diagonal spindles. Hematoxylin. x 2 400.
uranyl acetate and lead citrate and examined in a Hitachi electron microscope (HS-7s) at 50 kV. The term ‘kinetochore’ in the following text refers to the multilayered structure frequently observed in mammalian metaphase chromosomes. Since each of the two chromatids has its own kinetochore, the term has been used to designate a chromatid’s kinetochore if not otherwise mentioned.
RESULTS Fig. 1 shows examples of the multipolar metaphases found in the rat-kangaroo cell line Pt-Kl as seen in the light microscope. The metaphase chromosomes in fig. 1a form a Y-shaped equatorial plate of a tripolar spindle. In fig. lb, the equatorial plate portrays a T-shaped configuration and two of the
exhibiting (a) Yof spindles. The
four identifiable asters in this cell are located on the top of the T. As can be seen in such not too complex multipolar configurations, most of the chromosomesoccupy positions implying that they have a bipolar orientation. This is especially valid for chromosomesoriented in the middle and distal parts of the arms constituting the composite equatorial plate. The chromosomes in the central part of the composite equatorial plate, however, occupy peripheral positions of neighboring spindles (fig. 1a, b) as well as central positions of eventual diagonal spindles (fig. 1b). That such chromosomes might have multipolar connections was found
Fig. 2. Ultrastructure of kinetochores showing multipolar orientation. The multilayered appearance of the kinetochore is anparent. Usuallv the kinetochore is bent at a certain uoint so that two nearly equal (upper kinetochore (i) and the en&e fig. (b)); or unequal (lower kinetochore (a>) portions are oriented in different directions, as is also evident from the direction of the kinetochore microtubules (solid arrows). The kinetochore sometimes exhibits a circular appearance (c). Continuous microtubules can be seen in the vicinity of the kinetochore region (dashed arrows in b and c). The dashed arrow in (a) denotes an area where microtubules cross each other. a, x 32 500; b, c, x 47 300. Exptl
Cell Res 91 (1975)
Multipolar
Exptl
kinetochores
Cell
59
Res 91 (1975)
60
W. K. Heneen
stances, the kinetochore was usually sharply bent at one site so that halves or unequal parts faced different directions (fig. 2a, b). This bending does not affect the kinetochore multilayered organization. All kinetochore layers are bent at corresponding points and continue to be parallel. That the two kinetochore portions are oriented to two different poles is ascertained from the linear arrangement of the kinetochore microtubules which infers the exact direction of the two poles. The angle at the bending point of the kinetoFig. 3. An elaboration of the mechanism of ‘autochores shown in fig. 2a and b was close to orientation’ during mitosis as described and depicted by tjstergren [8]. The length of the arrow is pro90”. Multipolar kinetochores with narrower portional to the attraction force to a specific pole and wider angles were also encountered. whereas arrow thickness symbolizes the portion of the kinetochore and kinetochore microtubules that Kinetochore microtubules were usually perare oriented to a given pole. pendicular to the kinetochore surface in sections depicting a clear multilayered organizato be the caseas documented ultrastructurally tion of the kinetochore (fig. 2a, b). below. Microtubules were found to extend in Prometaphase and metaphasekinetochores various directions in multipolar divisions in in rat-kangaroo cells exhibited a variety of which the component spindles were at variappearances when viewed in the electron ous angles in relation to each other. This is microscope which is in accordance with the shown in fig. 2a where microtubules from observations of Roos [13] in a similar ma- two kinetochores cross each other apparterial. Some aspects of the variability in ently without interfering with the achievekinetochore appearance will be dealt with ment of the kinetochore-to-pole state of equilibrium. elsewhere. The multilayered kinetochore shown in Metaphase kinetochores frequently disfig. 2c has a nearly rounded appearance with played a multilayered appearance. The multimicrotubules that extend in two directions. layered structure was either parallel, slightly convex or concave, at an angle, or shaped In fig. 2b and c, it is possible to differentiate as an arrowhead, in relation to the adjacent what seemsto represent continuous microtubules. These usually appear as curved or underlying chromatin. In the multipolar metaphases studied, the majority of the microtubules in the vicinity of the two bunkinetochores exhibited normal bipolar orien- dles of kinetochore microtubules that are tation. The microtubules of each sister ki- directed towards different poles. netochore were oriented to only one polar region. This was inferred from the nearly DISCUSSION parallel and linear arrangement of kinetochore microtubules. The present observations confirm and extend In some chromosomes, the kinetochore the observation of Granholm [14] who found regions showed a picture deviating from cur- a tripolar rat-kangaroo cell in which one rently observed appearances. In these in- kinetochore was oriented to two separate Exptl
Cell Res 91 (1975)
Multipolar poles whereas the sister kinetochore was oriented to the third polar region. Thus in multipolar mitosis, a chromosome at full metaphase might be oriented and have persistent microtubule connections with more than two poles. The orientation of the kinetochore and kinetochore microtubules seems to be determined by the position of the chromosomes on the complex equatorial plate also in relation to the spindles developed between the surrounding polar regions. That a major part of the metaphase kinetochore and its microtubules are oriented to a certain polar region need not interfere with the orientation of the minor part to another polar region, as long as the forces acting in these two directions are in equilibrium with the forces acting on the sister kinetochore being oriented to another pole or other poles. In general, the present findings are in accordance with and supports the theory of ‘auto-orientation’ or ‘orientation by pulling’ of Ostergren [8]. Orientation of kinetochores and kinetochore microtubules to more than one polar region might be a situation that also prevails during the early stages of chromosome orientation in bipolar divisions. It has been documented [9] that kinetochores have connections with the two poles during the first and second divisions of meiosis. It has also been demonstrated [IO] that during the transition between prophase to metaphase of mitosis, kinetochore microtubules radiate in different directions, not only towards polar regions but also towards kinetochores of neighboring chromosomes. Kinetochore appearance and orientation of kinetochore microtubules during the progression of mitosis in normal bipolar configurations needs further documentation at the ultrastructural level. If bipolar orientation of chromatid kinetochores is of general occurrence during the transition from prophase to metaphase, this will be a further
kinetochores
61
support for the theory of ‘auto-orientation’ [8]. ijstergren’s schematic drawings ([8], figs 3, 4) could consequently be expressed as depicted in fig. 3. As the processes of autoorientation progresses, the portion of the kinetochore and kinetochore microtubules oriented to the ‘wrong’ pole will decrease. In general by full metaphase, only ‘right’ connections will lead to the equilibrium state of the metaphase chromosomes on the equatorial plate. Such behaviour would explain the mode of chromosome orientation observed in rat-kangaroo and other mammalian cells in culture during the transitional stages between prophase and metaphase. During these stages, the chromosomes were oriented at the equator as well as on the sides of the spindle and in the vicinity of the polar regions before they finally get oriented at the equatorial plate by full metaphase [ 11, 151. Various positions of chromosomes on the spindle most likely reflect different degrees of kinetochore orientation in relation to the two poles. In bipolar mitosis, partial orientation to the ‘wrong’ pole would be expected to cease already before reaching full metaphase since the angle between the two poles is wide (1807, and the sister kinetochores with their microtubules oriented in exactly or nearly opposite directions, would block possible partial orientations to the ‘wrong’ poles. In the case of multipolar mitosis, however, smaller angles prevail between neighboring polar regions which would allow the persistence of a multipolar orientation of the kinetochore to such poles. That the kinetochore can be distinguished as two equal or unequal components that have microtubules oriented to two different poles is an indication that any point on the kinetochore surface could represent a site of kinetochore/microtubule interaction. Microtubules directed from a certain kinetochore Exptl
Cell Res 91 (1975)
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W. K. Heneen
towards different poles are not intermingled but appear as two separate bundles at the kinetochore surface. This might evince the integrity of the component spindles and that these do not merge with each other at the equatorial plate region. That microtubules cross each other at intermediate positions between equatorial plates and poles is an indication that spindles probably merge into each other at these positions. The majority of the multipolar divisions in the Pt-Kl cell line are most likely of bior multinuclear origin [l 1, 161. Component spindles in multipolar configurations could thus be between sister or non-sister polar regions. Consequently, the observed continuous microtubules could be between sister or non-sister poles. The final appearance of multipolar metaphase configurations is most likely a product of the processes of the spindleorganizing capacity of chromosomes and the interactions that are partly between kinetochores and sister or non-sister poles and partly between such poles. Orientation of a kinetochore to more than two polar regions has not been documented in the multipolar configurations studied. Such a situation probably occurs in rather complex multipolar mitoses that are composed of several polar regions and component spindles. It is of interest to determine the behaviour of chromosomes exhibiting multipolar modes of orientation at anaphase. Such stages have
Exptl
Cell Res 91 (1975)
not been observed as yet but it would be expected that a chromatid will move to the pole towards which a greater part of the kinetochore and a great number of microtubules are oriented. Orientation of equal kinetochore parts to two polar regions might lead to breakage during anaphase The author wishes to express his sincere thanks to Mrs Bodil Strombeck and Mrs Britt Lernius for estimable technical assistance, and to Professor Albert Levan for critical reading of the manuscript. This work was supported by grants from the Swedish Cancer Society and the John and Augusta Persson Foundation.
REFERENCES 1. Brinkley, B R & Stubblefield, E, Adv cell biol 1 (1970) 119. 2. Luykx, P, Int rev cytol, suppl. 2 (1970). 3. Bajer, A S & Mole-Bajer, J, Int rev cytol, suppl. 3 (1972). 4. Stubblefield, E, Int rev cytol 35 (1973) 1. 5. Jokelainen, P T, J ultrastruct res 19 (1967) 19. 6. Comings, D E & Okada, T A, Exptl cell res 67 (1971) 97. 7. Brinkley, B R & Stubblefield, E, Chromosoma 19 (1966) 28. 8. bstkrgren, G, Hereditas 37 (1951) 85. 9. Luvkx. P. Exotl cell res 39 (1965) 658. 10. Bajer, ‘A’S & Mole-Bajer,- J, Chromosoma 27 (1969) 448. 11. Heneen, W K, Chromosoma 29 (1970) 88. 12. Brinkley, B R, Murphy, P & Richardson, L C, J cell biol 35 (1967) 279. 13. Roos, U-P, Chromosoma 41 (1973) 195. 14. Granholm. N A. Abstr 11 ann meet Am sot cell biol (1971 j 109. ’ 15. Heneen, W K, Nichols, W W, Levan, A & Norrby, E, Hereditas 64 (1970) 53. 16. Heneen, W K, Hereditas 67 (1971) 221. Received June 5,1974 Revised version received September 19, 1974
Experimental Cell Research91 (1975) 57-62
KINETOCHORES
AND AND
MICROTUBULES CHROMOSOME
IN MULTIPOLAR
MITOSIS
ORIENTATION
W. K. HENEEN Institute of Genetics and The Wallenberg Laboratory, University of Lund, S-223 62 Lund, Sweden
SUMMARY The ultrastructure of the kinetochore and the orientation of kinetochore microtubules were studied in multipolar divisions of cultured rat-kangaroo cells (Pt-Kl). The metaphase kinetochore exhibited a lamellar structure and most of the chromosomes expressed a bipolar microtubule orientation. A multipolar kinetochore orientation was observed in some chromosomes. Equal or unequal portions of a chromatid’s kinetochore and a corresponding number of kinetochore microtubules may be oriented to two different poles. Curved or bent continuous microtubules were observed in the vicinity of chromosomes showing multipolar orientation. The findings are in accordance with bstergren’s theory of ‘auto-orientation’ [S]. It is speculated that orientation of a chromatid kinetochore to more than one pole might be a possible regular event during the process of chromosome orientation prior to full metaphase in bipolar mitosis.
The kinetochore organization as well as the dynamics of chromosome movement and orientation are major topics of interest when dealing with processes of mitosis and meiosis. The present knowledge in this area is based mainly on light microscopic, cinematographic and, more recently, electron microscopic observations [l-4]. Ultrastructural analysis elucidates important aspects such as the detailed appearance of kinetochores, kinetochore/microtubule interrelationships and directions of individual microtubules. Elucidation of these features could be instructive for an understanding of chromosome movement and orientation. The metaphase kinetochore in mammalian materials is characterized by its lamellar appearance which was interpreted to represent a disc [5, 61 or a filamentous structure [l, 4, 71. Regular bipolar divisions were used in the majority of the studies that
deal with the fine structure of the mitotic apparatus. In general, each of the two sister metaphase kinetochores has microtubule connections with only one pole. ‘Wrong’ or ‘forbidden’ connections between one sister kinetochore and two polar regions or between two sister kinetochores and one polar region have also been documented both in mitosis and meiosis [8-lo]. The aim of the present investigation was to find out about the appearance and orientation of kinetochores in multipolar mitosis. MATERIALS
AND METHODS
The rat-kangaroo (Potorous tridactvlisj cell line Pt-Kl was used. Multipolar divisions are ’ frequently encountered (13.1 X) in this cell line which is also characterized by its flat mitotic cells [II]. The stemline chromosome number is 11 ($!,2n = 12). Cells were grown in Falcon plastic flasks and fixed and embedded using the technique of Brinkley et al. [12]. Cells in desirable mitotic stages were cut using an LKB microtome. The sections were stained with Exptl Cell Res 91 (1975)
58
W. K. Heneen
Fig. I. Multipolar metaphases in cultured rat-kangaroo (Potorous triductylis) cells (Pt-Kl) shaped, or (b) T-shaped equatorial plates. Solid and dashed lines indicate directions dashed lines represent possible diagonal spindles. Hematoxylin. x 2 400.
uranyl acetate and lead citrate and examined in a Hitachi electron microscope (HS-7s) at 50 kV. The term ‘kinetochore’ in the following text refers to the multilayered structure frequently observed in mammalian metaphase chromosomes. Since each of the two chromatids has its own kinetochore, the term has been used to designate a chromatid’s kinetochore if not otherwise mentioned.
RESULTS Fig. 1 shows examples of the multipolar metaphases found in the rat-kangaroo cell line Pt-Kl as seen in the light microscope. The metaphase chromosomes in fig. 1a form a Y-shaped equatorial plate of a tripolar spindle. In fig. lb, the equatorial plate portrays a T-shaped configuration and two of the
exhibiting (a) Yof spindles. The
four identifiable asters in this cell are located on the top of the T. As can be seen in such not too complex multipolar configurations, most of the chromosomesoccupy positions implying that they have a bipolar orientation. This is especially valid for chromosomesoriented in the middle and distal parts of the arms constituting the composite equatorial plate. The chromosomes in the central part of the composite equatorial plate, however, occupy peripheral positions of neighboring spindles (fig. 1a, b) as well as central positions of eventual diagonal spindles (fig. 1b). That such chromosomes might have multipolar connections was found
Fig. 2. Ultrastructure of kinetochores showing multipolar orientation. The multilayered appearance of the kinetochore is anparent. Usuallv the kinetochore is bent at a certain uoint so that two nearly equal (upper kinetochore (i) and the en&e fig. (b)); or unequal (lower kinetochore (a>) portions are oriented in different directions, as is also evident from the direction of the kinetochore microtubules (solid arrows). The kinetochore sometimes exhibits a circular appearance (c). Continuous microtubules can be seen in the vicinity of the kinetochore region (dashed arrows in b and c). The dashed arrow in (a) denotes an area where microtubules cross each other. a, x 32 500; b, c, x 47 300. Exptl
Cell Res 91 (1975)
Multipolar
Exptl
kinetochores
Cell
59
Res 91 (1975)
60
W. K. Heneen
stances, the kinetochore was usually sharply bent at one site so that halves or unequal parts faced different directions (fig. 2a, b). This bending does not affect the kinetochore multilayered organization. All kinetochore layers are bent at corresponding points and continue to be parallel. That the two kinetochore portions are oriented to two different poles is ascertained from the linear arrangement of the kinetochore microtubules which infers the exact direction of the two poles. The angle at the bending point of the kinetoFig. 3. An elaboration of the mechanism of ‘autochores shown in fig. 2a and b was close to orientation’ during mitosis as described and depicted by tjstergren [8]. The length of the arrow is pro90”. Multipolar kinetochores with narrower portional to the attraction force to a specific pole and wider angles were also encountered. whereas arrow thickness symbolizes the portion of the kinetochore and kinetochore microtubules that Kinetochore microtubules were usually perare oriented to a given pole. pendicular to the kinetochore surface in sections depicting a clear multilayered organizato be the caseas documented ultrastructurally tion of the kinetochore (fig. 2a, b). below. Microtubules were found to extend in Prometaphase and metaphasekinetochores various directions in multipolar divisions in in rat-kangaroo cells exhibited a variety of which the component spindles were at variappearances when viewed in the electron ous angles in relation to each other. This is microscope which is in accordance with the shown in fig. 2a where microtubules from observations of Roos [13] in a similar ma- two kinetochores cross each other apparterial. Some aspects of the variability in ently without interfering with the achievekinetochore appearance will be dealt with ment of the kinetochore-to-pole state of equilibrium. elsewhere. The multilayered kinetochore shown in Metaphase kinetochores frequently disfig. 2c has a nearly rounded appearance with played a multilayered appearance. The multimicrotubules that extend in two directions. layered structure was either parallel, slightly convex or concave, at an angle, or shaped In fig. 2b and c, it is possible to differentiate as an arrowhead, in relation to the adjacent what seemsto represent continuous microtubules. These usually appear as curved or underlying chromatin. In the multipolar metaphases studied, the majority of the microtubules in the vicinity of the two bunkinetochores exhibited normal bipolar orien- dles of kinetochore microtubules that are tation. The microtubules of each sister ki- directed towards different poles. netochore were oriented to only one polar region. This was inferred from the nearly DISCUSSION parallel and linear arrangement of kinetochore microtubules. The present observations confirm and extend In some chromosomes, the kinetochore the observation of Granholm [14] who found regions showed a picture deviating from cur- a tripolar rat-kangaroo cell in which one rently observed appearances. In these in- kinetochore was oriented to two separate Exptl
Cell Res 91 (1975)
Multipolar poles whereas the sister kinetochore was oriented to the third polar region. Thus in multipolar mitosis, a chromosome at full metaphase might be oriented and have persistent microtubule connections with more than two poles. The orientation of the kinetochore and kinetochore microtubules seems to be determined by the position of the chromosomes on the complex equatorial plate also in relation to the spindles developed between the surrounding polar regions. That a major part of the metaphase kinetochore and its microtubules are oriented to a certain polar region need not interfere with the orientation of the minor part to another polar region, as long as the forces acting in these two directions are in equilibrium with the forces acting on the sister kinetochore being oriented to another pole or other poles. In general, the present findings are in accordance with and supports the theory of ‘auto-orientation’ or ‘orientation by pulling’ of Ostergren [8]. Orientation of kinetochores and kinetochore microtubules to more than one polar region might be a situation that also prevails during the early stages of chromosome orientation in bipolar divisions. It has been documented [9] that kinetochores have connections with the two poles during the first and second divisions of meiosis. It has also been demonstrated [IO] that during the transition between prophase to metaphase of mitosis, kinetochore microtubules radiate in different directions, not only towards polar regions but also towards kinetochores of neighboring chromosomes. Kinetochore appearance and orientation of kinetochore microtubules during the progression of mitosis in normal bipolar configurations needs further documentation at the ultrastructural level. If bipolar orientation of chromatid kinetochores is of general occurrence during the transition from prophase to metaphase, this will be a further
kinetochores
61
support for the theory of ‘auto-orientation’ [8]. ijstergren’s schematic drawings ([8], figs 3, 4) could consequently be expressed as depicted in fig. 3. As the processes of autoorientation progresses, the portion of the kinetochore and kinetochore microtubules oriented to the ‘wrong’ pole will decrease. In general by full metaphase, only ‘right’ connections will lead to the equilibrium state of the metaphase chromosomes on the equatorial plate. Such behaviour would explain the mode of chromosome orientation observed in rat-kangaroo and other mammalian cells in culture during the transitional stages between prophase and metaphase. During these stages, the chromosomes were oriented at the equator as well as on the sides of the spindle and in the vicinity of the polar regions before they finally get oriented at the equatorial plate by full metaphase [ 11, 151. Various positions of chromosomes on the spindle most likely reflect different degrees of kinetochore orientation in relation to the two poles. In bipolar mitosis, partial orientation to the ‘wrong’ pole would be expected to cease already before reaching full metaphase since the angle between the two poles is wide (1807, and the sister kinetochores with their microtubules oriented in exactly or nearly opposite directions, would block possible partial orientations to the ‘wrong’ poles. In the case of multipolar mitosis, however, smaller angles prevail between neighboring polar regions which would allow the persistence of a multipolar orientation of the kinetochore to such poles. That the kinetochore can be distinguished as two equal or unequal components that have microtubules oriented to two different poles is an indication that any point on the kinetochore surface could represent a site of kinetochore/microtubule interaction. Microtubules directed from a certain kinetochore Exptl
Cell Res 91 (1975)
62
W. K. Heneen
towards different poles are not intermingled but appear as two separate bundles at the kinetochore surface. This might evince the integrity of the component spindles and that these do not merge with each other at the equatorial plate region. That microtubules cross each other at intermediate positions between equatorial plates and poles is an indication that spindles probably merge into each other at these positions. The majority of the multipolar divisions in the Pt-Kl cell line are most likely of bior multinuclear origin [l 1, 161. Component spindles in multipolar configurations could thus be between sister or non-sister polar regions. Consequently, the observed continuous microtubules could be between sister or non-sister poles. The final appearance of multipolar metaphase configurations is most likely a product of the processes of the spindleorganizing capacity of chromosomes and the interactions that are partly between kinetochores and sister or non-sister poles and partly between such poles. Orientation of a kinetochore to more than two polar regions has not been documented in the multipolar configurations studied. Such a situation probably occurs in rather complex multipolar mitoses that are composed of several polar regions and component spindles. It is of interest to determine the behaviour of chromosomes exhibiting multipolar modes of orientation at anaphase. Such stages have
Exptl
Cell Res 91 (1975)
not been observed as yet but it would be expected that a chromatid will move to the pole towards which a greater part of the kinetochore and a great number of microtubules are oriented. Orientation of equal kinetochore parts to two polar regions might lead to breakage during anaphase The author wishes to express his sincere thanks to Mrs Bodil Strombeck and Mrs Britt Lernius for estimable technical assistance, and to Professor Albert Levan for critical reading of the manuscript. This work was supported by grants from the Swedish Cancer Society and the John and Augusta Persson Foundation.
REFERENCES 1. Brinkley, B R & Stubblefield, E, Adv cell biol 1 (1970) 119. 2. Luykx, P, Int rev cytol, suppl. 2 (1970). 3. Bajer, A S & Mole-Bajer, J, Int rev cytol, suppl. 3 (1972). 4. Stubblefield, E, Int rev cytol 35 (1973) 1. 5. Jokelainen, P T, J ultrastruct res 19 (1967) 19. 6. Comings, D E & Okada, T A, Exptl cell res 67 (1971) 97. 7. Brinkley, B R & Stubblefield, E, Chromosoma 19 (1966) 28. 8. bstkrgren, G, Hereditas 37 (1951) 85. 9. Luvkx. P. Exotl cell res 39 (1965) 658. 10. Bajer, ‘A’S & Mole-Bajer,- J, Chromosoma 27 (1969) 448. 11. Heneen, W K, Chromosoma 29 (1970) 88. 12. Brinkley, B R, Murphy, P & Richardson, L C, J cell biol 35 (1967) 279. 13. Roos, U-P, Chromosoma 41 (1973) 195. 14. Granholm. N A. Abstr 11 ann meet Am sot cell biol (1971 j 109. ’ 15. Heneen, W K, Nichols, W W, Levan, A & Norrby, E, Hereditas 64 (1970) 53. 16. Heneen, W K, Hereditas 67 (1971) 221. Received June 5,1974 Revised version received September 19, 1974
