THE KINETIC POLARITIES OF SPINDLE …jcs.biologists.org/content/joces/79/1/39.full.pdf · (assembly) ends of treadmilling microtubules (Farrell & Wilson, 1984), ... chromosomal spindle

J. Cell Sri. 79, 39-65 (1985) 39Printed in Great Britain © The Company of Biologists Limited 1985

THE KINETIC POLARITIES OF SPINDLEMICROTUBULES IN VIVO, IN CRANE-FLYSPERMATOCYTES. II. KINETOCHOREMICROTUBULES IN NON-TREATED SPINDLES

B. BARBARA CZABAN AND ARTHUR FORER

Biology Department, York University, Downsvievi, Ontario, Canada M3J 1P3

SUMMARY

We determined the kinetic polarities of chromosomal 9pindle fibre microtubules in vivo: eitherthe kinetochore or pole ends of chromosomal spindle fibres were irradiated with near-ultravioletlight to prevent depolymerization by colcemid. Irradiations began either just before or just aftercolcemid addition; cells were continually irradiated and continuously immersed in colcemid.Irradiations of kinetochore ends of chromosomal spindle fibres prevented depolymerization;irradiations of pole ends did not. Therefore, since colcemid acts by binding to the 'on' (assembly)ends of microtubules, the on ends of chromosomal spindle fibre microtubules are at thekinetochores. That is, in untreated chromosomal spindle fibres in vivo tubulin monomers add tokinetochore microtubules at the kinetochore ends.

Tubulin diffused from the irradiation sites: irradiations of the cytoplasm sometimes preventeddepolymerization of chromosomal spindle fibres. Prevention of chromosomal spindle fibredepolymerization was dependent on the distance of the irradiated region from the nearestchromosome; the longer the distance the less likely was it that the irradiation preventeddepolymerization. On the other hand, prevention of chromosomal spindle fibre depolymerizationwas not dependent on the distance from the irradiated spot to the nearer pole. This analysis, too, weargue, strongly suggests that the kinetochore ends of the chromosomal spindle fibres are the onends.

INTRODUCTION

Microtubules are important components of the mitotic spindle. To helpunderstand how spindle microtubules might function it is important to know thestructural and kinetic polarities of spindle microtubules, of both the chromosomalspindle fibre (or kinetochore) microtubules (those that attach to the chromosome andextend to the pole) and the interpolar microtubules (those that extend from one poletowards the other but end freely before reaching the other pole). The structuralpolarities are known: all chromosomal and interpolar microtubules in a given half-spindle (between the chromosomes and a pole) have the same structural polarities;further, microtubules in opposite half-spindles have opposite structural polarities(Mclntosh & Euteneur, 1984; Telzer & Haimo, 1981).

The kinetic polarities of spindle microtubules are not known, however, and thereare several different hypotheses about the kinetic polarities of spindle microtubules(e.g. see Margolis, 1978; Goode, 1981; Bergen, Kuriyama & Borisy, 1980; Summers

Key words: kinetic polarities, spindle microtubules, colcemid.

40 B. B. Czaban and A. Forer

& Kirschner, 1979; Tippit, Pickett-Heaps & Leslie, 1980; De Brabander, 1982;Rieder, 1982). Kinetic polarities of spindle microtubules often are assumed (e.g. seePickett-Heaps, Tippit & Porter, 1982; Mclntosh, 1984), on the basis of the knownrelationship between kinetic and structural polarities of free microtubules in vitro(e.g. see Bergen & Borisy, 1980; Summers & Kirschner, 1979). But external factors(such as local conditions or 'capping' proteins) can influence kinetic polarities, so thekinetic polarities of spindle microtubules in vivo may differ from those predictedfrom the structural polarities: it is necessary to determine the kinetic polaritiesindependently of structural polarities.

The kinetic polarities of chromosomal spindle fibre microtubules were determinedwhen the microtubules re-formed after recovery from colcemid treatment (Czaban &Forer, 1985). In those experiments we added colcemid to spermatocytes and afterspindle birefringence was no longer detectable we irradiated small regions of the cellswith a 'microbeam' of near-ultraviolet light to convert the colcemid to lumicolcemidin that local region; since lumicolcemid does not bind to tubulin, the microbeamirradiations created free tubulin in the irradiated region and allowed birefringentchromosomal spindle fibres to be re-formed. By irradiating different regions of thecell and studying the re-formation of spindle birefringence we deduced: (a) that thechromosomal spindle fibres were organized by the chromosomes; and (b) thattubulin monomers added onto the chromosomal spindle fibres at the kinetochores.The latter is because the chromosomal spindle fibres grew (lengthened) from thechromosomes out past the edges of the irradiated spots through distances that freetubulin would not be able to diffuse without interacting with colcemid.

Experiments in which cells were studied after recovery from colcemid treatment(e.g. see Witt, Ris & Borisy, 1980, 1981) have been criticized, however. It has beenargued that the results are artefacts of pretreatment with colcemid, due perhapsto special circumstances or to remnants of microtubules remaining near thekinetochores, and that in fact all spindle fibre microtubules in vivo are organized bythe poles (Pickett-Heaps & Tippit, 1978; Tippit et al. 1980; Pickett-Heaps et al.1982). It has been argued that while kinetochores can act as nucleating centres formicrotubules under some conditions, kinetochores in vivo capture microtubulesnucleated by the poles (Rieder, 1982). To test these criticisms we have determinedthe kinetic polarities of chromosomal spindle fibre microtubules in vivo, without pre-treatment with colcemid or other depolymerizing drugs.

In our experiments we irradiated different areas of crane-fly spermatocytes with amicrobeam of near-ultraviolet light; irradiations started just before or just aftercolcemid was added and continued thereafter. Since the irradiation converts thecolcemid in the irradiated spot to lumicolcemid, the irradiation should preventdisappearance of birefringence if the irradiated region contains the ends of themicrotubules that colcemid binds to. Since colcemid binds preferentially to the 'on'(assembly) ends of treadmilling microtubules (Farrell & Wilson, 1984), i.e. to thefast-polymerizing ends of polymerizing microtubules (Bergen & Borisy, 1980),irradiations of this kind identify the on ends of the microtubules in the irradiatedspindle fibre. In this way we have deduced that the on ends of non-treated

Microtubide kinetic polarities in vivo 41

chromosomal spindle fibre microtubules are at the kinetochores, in agreement withour result on spindle fibres that re-formed after colcemid treatment (Czaban &Forer, 1985).

MATERIALS AND METHODSDetails of the animals, cells and methods are given in the preceding paper (Czaban & Forer,

1985). In brief, crane-fly spermatocytes were prepared in smear or clot preparations. For a smearpreparation, a whole testis was immersed in a drop of colcemid solution under halocarbon oil; thenthe testis was pierced, the cells mixed with the colcemid, and the cells smeared into a monolayerunder the oil. Cells for study were located and irradiated within 5 min of the initial immersion incolcemid. In clot preparations cells were held in place in a fibrin clot, under oil; then the oil wasreplaced with Ringer's solution and a cell located for study; then irradiation was started, 2min ormore before addition of colcemid, and then colcemid was added to replace the Ringer's solution.While chromosomal spindle fibre birefringence was not altered before the irradiation started, ineither smear or clot preparation, the smear preparation cells were already in colcemid before theirradiation started; it is possible that colcemid affected these cells before the irradiation started. Onthe other hand, clot preparation cells were irradiated before colcemid was added; colcemid couldnot have affected those cells before the irradiation started.

The microbeam spot was visible during the irradiation because violet light was transmittedtogether with the broad band of near-ultraviolet light that included 360nm wavelength light; inmany cases the spot was moved around to follow the part of the cell that was being irradiated (e.g. ifthe irradiated chromosome moved). For each irradiation we monitored the dose rate at themicrobeam aperture (a slit) and used a measured transmission coefficient to calculate the dose rate(of 363 nm wavelength light) incident on the cell. During the irradiation cells were observed on atelevision monitor (via video-enhanced bright-field microscopy); cells were also studied usingpolarization microscopy or phase-contrast microscopy, but only briefly and intermittently sincethese observations reduced the intensity of the microbeam. The images were recorded on videotape and 35 mm film.

RESULTS

Our experimental design was basically to irradiate different parts of a cell with360 nm light to see if the irradiation prevented the disappearance of birefringencethat colcemid otherwise would induce; in these 'prevention' experiments irradiationscontinued until either the birefringence was no longer detectable or the cell enteredanaphase. Clearly, then, to see if the irradiations were 'successful' in preventingloss of birefringence we need to know how long it takes for colcemid to causedisappearance of birefringence in non-irradiated cells and we need to compare thiswith the length of time that birefringence remained in cells that were irradiated. Inthe preceding article (Czaban & Forer, 1985) we described the effects of colcemid onnormal cells, and we described how the effects depend on concentration of colcemidand stage of meiosis. For reasons given in that article the experiments described inthis article were done exclusively with cells in prometaphase (as judged by thechromosomes being spread out along the spindle axis) and with colcemid at aconcentration of 10~6M.

Before describing how long it takes colcemid to cause spindle birefringence todisappear from non-irradiated cells it is relevant to review briefly the effect ofcolcemid on prometaphase spindles (Czaban & Forer, 1985). The first change seen


8o

Iz

20

18

16

14

12

108

6

4

2 r

A

110 20 30 40 50 60 70 80 90 100 110

Time birefringence remained (min)

20

18

16

= 148•5 12| i o

1 86

4

2

n

B

1—110 20 30 40 50 60 70 80 90 100110

Time birefringence remained (min)

Fig. 1. The times that birefringence remained: A, in non-irradiated cells; and B, aftersuccessful prevention irradiations. A. The number of cells (ordinate) versus the time (inmin) until spindle birefringence was no longer detectable (abscissa). B. The number ofcells (ordinate) versus the time (in min) between the start of irradiation and onset ofanaphase (abscissa), for all cells in which prevention irradiations were scored as successfulusing criteria discussed in the text.

after addition of colcemid is loss of asters. After this the chromosomal spindle fibresdo not focus to the poles, but rather the spindle becomes barrel-shaped. Then thechromosomal spindle fibres shorten until the spindle disappears.

We measured how long it took for spindle birefringence to disappear fromprometaphase cells. (This experiment comprises a series of cells separate fromthose described in the preceding article (Czaban & Forer, 1985).) Birefringence

Microtubule kinetic polarities in vivo 43

A. Whole cell B. 3-Chromosome

c. 2-Chromosome D. 1-Chromosome

Fig. 2. Diagrammatic representations of the regions of cells that were irradiated inprevention experiments. A, asters; SF, spindle fibres; P, poles; C, chromosomes.

disappeared after 29-2 ±7*2 min (± standard deviation) after 10~6M-colcemid wasadded to 37 non-irradiated prometaphase cells (Fig. 1A).

In those prevention experiments scored as successful, birefringent spindle fibresremained for at least 30 min (between the start of the irradiation and the beginning ofanaphase), with a range of 32-109 min (Fig. 1B). When birefringence disappeared in


less than 30min the experiment was scored as not successful. When birefringencehad not disappeared but anaphase started before 30min of irradiation, the cell wasscored as ambiguous and the data were not used.

An additional criterion was also used to score prevention irradiations as successful,namely to look at nearby cells. Cells were irradiated either until spindle birefringencedisappeared or until anaphase started. When anaphase started we stopped theirradiation and immediately studied neighbouring cells to see if birefringent spindleswere present in the nearby cells. Birefringent spindle fibres were not seen inneighbouring cells in prevention experiments scored as successful; rather, there wereclumped chromosomes, indicating that the spindles had disappeared from the non-irradiated cells (see descriptions (Czaban & Forer, 1985)). On the other hand, therewere a few cells in which anaphase occurred 32-40 min after the start of irradiationthat would have been scored as successful 'prevention' experiments but that ratherwere scored as ambiguous and discarded because a nearby cell also had birefringentspindle fibres and was in anaphase.

We now describe the successful irradiations: these were of whole cells or ofportions of cells, as follows.

Whole-cell irradiations

Irradiations of whole cells (Fig. 2A) prevented loss of birefringence in three cells(out of three attempts), all of which were in smear preparations (Table 1). In thesecells the birefringent chromosomal spindle fibres remained during the entireirradiation period but the general appearance of the spindle changed, much as itdid in non-irradiated cells. First, the asters disappeared, within 10-20 min aftercolcemid addition; then the remaining spindle fibres lost their focus to the poles andthe spindle became barrel-shaped. After this, there was sometimes shortening of thechromosomal spindle fibres, sometimes not, and sometimes after shortening therewas re-elongation. (In each cell, all three chromosomal spindle fibres shortened orelongated at the same time.) In one cell the chromosomal spindle fibres shortened34% from their original length, and then they lengthened again by 22% from theirshortest length; in a second cell the fibres increased 11 % in length without any initialshrinkage; and in the third cell the spindle did not change in length. Nonetheless,although the spindles sometimes changed somewhat, whole-cell irradiationsprevented the loss of spindle birefringence.

Partial-cell irradiations

Various areas within cells were irradiated to see if the irradiations would preventloss of spindle birefringence. In these prevention experiments the sizes and shapes ofthe irradiation spots were adjusted to fit the geometry of the cell being irradiated. Inall cases the irradiating spot was as square as possible, rather than a long thinrectangle.

Irradiations of chromosomes. Irradiated areas included either three chromosomes,two chromosomes, or one chromosome (Fig. 2B-D), in total areas ranging from63 /um2 to 394 fxm2 (three chromosomes), 45 jUm2 to 128 /an2 (two chromosomes), or


42/im2 to 81 ^m2 (one chromosome). The effective irradiated areas were the totalareas of the irradiation spots minus the areas of the chromosomes (each chromosomehad an area of about 10—15 /im2).

Three chromosomes were irradiated in 19 cells, and, as summarized in Table 1,each irradiation was successful: spindle fibres remained associated with eachchromosome throughout the entire irradiation period (Figs 5-7). In 12 'other' cellsthe chromosomes shifted their relative positions during irradiation; we did not

Table 1. Success rates of prevention experiments

Irradiation site

Whole-cell3-chromosome2-chromosome1-chromosomeOther

Cells withbirefringence remaining(fraction of total number

of attempts)

3/319/1913/172/96/12

Fraction of successfulprevention irradiations

Cell preparations:

Smears Clots

3/311/114/72/92/2

08/89/100

4/10

n- 5

n - 4 n - 1 n - 2 n = l n - 1

n - 2 n - 1

0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10

Area xlO2 (fan2)

n - 3

100

80

60

40

20

0

n-2 n-1 n-2

/ n-2A n - 1 2

In = 21

•In -15

n - 1 n — 1 B

n = 3

—• • II •0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10

AreaxlO2 (jan2)

Fig. 3. The percentage of successful prevention irradiations (ordinate) versus the areaof the irradiated region (abscissa). A. Cells in smear preparations. B. Cells in clotpreparations as well as cells in smear preparations (i.e. those in A), n is the number of cellsirradiated.


100

*

60

40

20

/in-8

=•10

n ** 1 / ! • 1 n • 3 n —1 A

n - 4

0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-« 8-9 9-10 >10

Energy flux X10~20*W)

3

100

80

60

40

20

n

n

/ "

Yn-TA

Tn-U

' -

- 3 n - 1 n - 1

n - 3

•12

—• It—n - 1

— H—

B

n - 4

0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10

Energy fluxx 10~2 (jiW)

>10

Fig. 4. The percentage of successful prevention irradiations (ordinate) versus the energyflux of each irradiation (abscissa). A. Cells in smear preparations. B. Cells in clotpreparations as well as cells in smear preparations (i.e. those in A), n is the number of cellsirradiated.

change the shape of the spot, so in these experiments some chromosomes moved inand out of the irradiated spots, thereby causing different combinations of chromo-somes to be irradiated. Six of the other prevention irradiations were successful(Table 1). One chromosome was irradiated in nine cells and two chromosomes wereirradiated in 17 cells; 15 of these successfully prevented loss of spindle birefringence(Table 1).

When partial-cell prevention experiments were not successful, spindle bi-refringence disappeared exactly as in cells that were not irradiated. When partial-cellprevention experiments were successful, spindles often changed just as when wholecells were irradiated. That is to say, during successful prevention irradiations astersoften disappeared and spindles became barrel-shaped, after which chromosomalspindle fibres sometimes shortened in length and sometimes did not, and theshortened spindle fibres sometimes elongated again (e.g. see Figs 5-8). In particular,chromosomal spindle fibres shortened in about 70% of the successful partial-cellprevention irradiations and elongated again in about 40 % of those cells in which theyhad shortened (Table 2): chromosomal spindle fibres that shortened duringsuccessful prevention irradiations did so by up to 80 % of the initial spindle fibrelength of 10-12/im (Table 3).

Microtutnde kinetic polarities in vivo 47

Fig. 5. Prevention irradiation of three autosomes, in a prometaphase cell in a clotpreparation. The three autosomes are indicated by bars (in A). After the astersdisappeared the spindle became barrel-shaped (D—F); the arrows in F indicate thedirections of the autosomal chromosomal spindle fibres and those in O indicate the threepairs of separating autosomal half-bivalents (in anaphase). The irradiation began at1-0 min and colcemid was added at 5-0min; anaphase started at 44min. A,D,G. Phase-contrast microscopy. B,C,E,F,H. Polarization microscopy. Times of the photographs:A, 3-0min; B, 4-0min; C, 22-0min; D, 23-Omin; E, 32-0min; F, 45-0min; G, 46-0min;H, 52-0 min. X937.

Fig. 6. Successful prevention irradiation of three chromosomes in a cell in a clotpreparation, illustrating diverging chromosomal spindle fibres (arrows in D) after theasters disappeared. The light rectangle in B is the image of the irradiated spot. Theirradiations began at 10-0 min and colcemid was added at ll-0min. A,C,D. Polarizationmicroscopy; B, bright-field microscopy. Times: A, 7-3min; B, 13-0min; c, 23-Omin;D, 49-0min. X785.


Colcemid can diffuse into and out of the irradiated spot, as discussed previously(Czaban & Forer, 1985). Thus the areas of the irradiating spots were important indetermining the success of prevention experiments: the larger the area the morelikely that the irradiation prevented the loss of spindle birefringence (Fig. 3).Indeed, as discussed previously (Czaban & Forer, 1985), one really should considerthe energy flux, i.e. the area of the irradiated spot multiplied by the dose rate (energyper time per area) of each individual irradiation: the success of a given experimentwas directly proportional to the energy flux of the irradiation, prevention irradiationsbeing successful 100% of the time when energy fluxes were >3xlO~2/zW (Fig. 4).

Free tubulin can diffuse out of the irradiated spot: prevention irradiations of oneor two chromosomes were successful in 15 cells but in 13 of these spindle fibresremained associated with each and every one of the three autosomes, throughout theexperiments, despite the fact that not all chromosomes were in the irradiated area(Figs 9, 10). The birefringent spindle fibres associated with the non-irradiated

J

Fig. 7. Successful prevention irradiation of three chromosomes in a cell in a smearpreparation. The spindle fibres lengthened after having first shortened. The lightrectangle in c is the irradiated spot. Colcemid was added at 0 min and irradiation began at5-0min; anaphase started at 75-0min. A,H,J. Phase-contrast microscopy; B,D-G,I ,K,L,polarization microscopy; c, bright-field microscopy. Times: A, 4-3min; B, 9-0min; c,12-5min; D, 16-0min; E, 23-0min; F, 35-0min; G, 37-0min; H, 48-0min; I, 64-0min; J,76-0min; K, 82-0min; L, 98-0min. X785.


chromosome disappeared in only two of the IS successful experiments (Table 4; Figs11-12). (In one other cell the chromosomal spindle fibres of the non-irradiatedchromosome were distinctly less birefringent (as judged by eye) than thechromosomal spindle fibres of the irradiated chromosomes.) Likewise, in the othercells at least one chromosome was in and out of the irradiated spot yet birefringentspindle fibres were associated with each of the three chromosomes throughout the

Fig. 8. Successful prevention irradiation of chromosomes in which some chromosomesmoved in and out of the irradiated spot, in a cell in a clot preparation. The light rectanglein C is the irradiated spot. Two autosomes originally were irradiated (c) but the third latermoved into the irradiated spot. Chromosomal spindle fibres remained associated with allthree autosomes (arrows), in a barrel-shaped spindle. The irradiation began at 6-0 minand colcemid was added at 8-0 min. Anaphase started at 54-0 min. A,F,H. Phase-contrastmicroscopy; B,D,E,G, polarization microscopy; C, bright-field microscopy. Times:A, Omin; B, 3-0min; c, 10-0min; D, 20-0min; E, 31-0min; F, 33-0min; G, 52-0min;H, 56-0 min. X937.

Table 2. Summary of partial-cell prevention irradiations of chromosomesTotal number of successful partial-cell prevention irradiations: 40

N u m b e r of successfulprevention irradiations inwhich spindle fibres did notshorten

Of the 13, the number inwhich the spindle fibreselongated to longer thantheir initial length

13 27 Number of successfulprevention irradiations inwhich spindle fibresshortened during theirradiation

TZ Of the 27, the number inwhich spindle fibreselongated again during theirradiation period


Fig. 9. Successful prevention irradiation of two chromosomes in a cell in a smearpreparation. The light rectangle in C is the irradiated spot. The non-irradiatedchromosome is indicated by a bar (c). Birefringent chromosomal spindle fibres remainedassociated with all three chromosomes. Colcemid was added at 0 min and the irradiationbegan at 6-0min; anaphase began at 76-0min. A,F,L. Phase-contrast microscopy;B,D,E,G— K, polarization microscopy; C, bright-field microscopy. Times: A, 4-0min; B,5-0min; c, l l-0min; D, 17-0min; E, 25-0min; F, 32-0min; G, 33-5min; H, 44-0min; I,62-0min; J, 72-0min; K, 78-0min; L, 82-0min. X785.

Table 3. Amount of spindle shortening during prevention irradiations

Number of cells with the statedpercentage of shortening (%)

Irradiation site

Whole-cell3-chromosome2-chromosome1-chromosomeOthers

Total numberof cells

0

28203

15

10-20

03400

7

21-40

12110

5

41-60

05613

IS

61-80

01000

1

100

00476

17

Microtubule kinetic polarities in vivo £1

experiment (Fig. 8). Since spindle fibres associated with chromosomes outside theirradiated areas did not depolymerize, free tubulin must have diffused from theirradiated areas to one of the two ends of the spindle fibres outside the irradiated area,either to the other chromosomes or to the poles.

Fig. 10. Successful prevention irradiation of one chromosome, in a cell in a smearpreparation. The light rectangle in B is the irradiated spot. The two non-irradiatedchromosomes are indicated by bars (B). Birefringent chromosomal spindle fibresremained associated with all three chromosomes. The spindle fibres first shortened andthen lengthened. Colcemid was added at Omin and the irradiation began at 5*5 min;anaphase started at 73-0min. A,D,F. Polarization microscopy; c,E,G,H, phase-contrastmicroscopy; B, bright-field microscopy. Times: A, 4-0min; B, 15-0min; C, 16-5min;D, 22-0min; E, 37-0min; F, 62-0min; G, 64-0min; H, 87-0min. X785.

Table 4. Number of birefringent spindle fibres that remained in successful preventionexperiments

Irradiation site

Whole-cell3-chromosome2-chromosome1-chromosomeOther

Successfulpreventions(fraction of

total numberof attempts)

3/319/1913/172/96/12

Number of cellswith three spindlefibres remaining

3191126

Number of cellswith no spindle fibres

remaining with theunirradiated chromosome

0Q200

B. B. Czaban and A. Forer

Fig. 11. Successful prevention irradiation of two chromosomes in a cell in a clotpreparation. The light rectangle in C is the irradiated spot. The non-irradiatedchromosome is indicated by a bar. Birefringent chromosomal spindle fibres remainedassociated with the two chromosomes that were irradiated but not with the one that wasnot irradiated. At anaphase the irradiated autosomes disjoined into poleward moving half-bivalents whereas the non-irradiated autosome remained as a bivalent (E) . The irradiationbegan at 3-5 min and colcemid was added at 5-0min; anaphase started at 43-0min. A,E.Phase-contrast microscopy; B,D, polarization microscopy; C, bright-field microscopy.Times: A, l-0min; B, 2-0min; C, 7-0min; D, 35-0min; E, 52-0min. X937.

Free tubulin most probably diffused to the chromosomes, not the poles, asindicated by comparison of two sets of distances: (1) the distances between theirradiating spots and the poles; and (2) the distances in the colcemid-treated cell thattubulin could diffuse through and still remain biologically active. Since the distancesbetween spots and poles are larger than the diffusion distance then the tubulin cannothave diffused to the poles but must have diffused to the kinetochores, as follows.

The distance between each irradiating spot and the nearer pole was measured fromthe photographs (or video images) of each cell. Some spindles shortened during theirradiation and others did not (Table 2). For each cell in which spindle fibresshortened during the irradiation, the distance was measured between the spot andthe closer spindle pole at the shortest spindle fibre length; these minimum distances(between irradiated spots and poles) ranged from 0/am to 8-4/im (Fig. 13A). For cellsin which spindle fibres did not shorten during the irradiation, the distances rangedfrom 0-5 /zm to 160 fitn (Fig. 13c). These two sets of data are pooled in Fig. 13D, toillustrate the set of all minimum distances. Thus, if diffusion to the pole accounts forprevention of birefringence, tubulin must be able to diffuse these minimum distancesand remain biologically active. Indeed, tubulin must be able to diffuse farther thanthese distances, for as the spindle fibres that shortened elongated again (Fig. 13B), oras the non-shortened spindle fibres elongated or kept of constant length, the tubulinmust continue to be able to diffuse to the polar ends if those are indeed the on ends.


These distances, the maximum distances (to the nearer pole) that the tubulin mustbe able to diffuse through if the polar ends are the on ends, ranged from 0/im to16-8/im(Fig. 13E).

The distance that tubulin can diffuse through was determined empirically: areas ofthe cytoplasm were irradiated at varying distances from the spindle in attempts toprevent loss of spindle birefringence. For each irradiation we measured the distancebetween the irradiated spot and the closer pole, and between the spot and the closestchromosome (Fig. 14). (The distances to the other two chromosomes were alwaysgreater than the distances to the closest chromosomes.) A successful diffusionexperiment was one in which spindle fibres associated with the closest chromosomedid not disappear throughout the experiment.

Fig. 12. Successful prevention irradiation of two chromosomes in a cell in a clotpreparation. The light rectangle in (C,F) is the irradiated spot. (The dark line across therectangle in F is due to noise on the videotape.) The non-irradiated chromosome isindicated by a bar. The birefringent chromosomal spindle fibres associated with the twoirradiated chromosomes remained but those associated with the non-irradiated chromo-some disappeared. The irradiated chromosomes disjoined and the resultant half-bivalentsmoved polewards (in anaphase) whereas the non-irradiated autosome remained non-disjoined, at the equator (H,J) . The irradiation began at 16-0 min and colcemid was addedat 17-0 min; anaphase started at 50-0 min. A,E,H,J. Phase-contrast microscopy; B,D,G,I,polarization microscopy; C,F, bright-field microscopy. Times: A, 10-0min; B, 13-0min;C, 20-0min; D, 31-0min; E, 38-0min; F, 39-Smin; G, 45-0min; H, 51-0min; i, 56-0min;J, 60-0min. X937.


Diffusion experiments were successful in five out of 10 cells. (During successfuldiffusion experiments spindle birefringence changed in exactly the same manner asin experiments in which chromosomes were directly irradiated, that is, astersdisappeared and the birefringent chromosomal spindle fibres sometimes shortenedand sometimes did not (e.g. see Fig. 15).) The success of any diffusion irradiation inpreventing loss of spindle birefringence depended on the distance between theirradiating spot and the nearest chromosome, the farther the chromosome the lesslikely that spindle fibre birefringence remained (Fig. 16A); there were no successfuldiffusion irradiations for distances above 4-5 [un, consistent with the diffusiondistances determined during re-formation of spindle fibres after colcemid treatment(fig. 18 of Czaban & Forer, 1985), as illustrated by pooling the two sets of data (Fig.16B). On the other hand, the success of a diffusion irradiation in preventing loss ofspindle birefringence did not seem to depend on the distance between the spot andnearer pole (Fig. 17): some closer spots were unsuccessful, whereas some fartherspots were successful. Thus, these data strongly suggest that tubulin diffuses fromthe irradiated spot in the cytoplasm to the nearest chromosome, for distances up to4—5 /im. Indeed, in those cells in which one or two chromosomes were irradiated butall three sets of autosomal spindle fibres remained, the distances between the spotsand the nearest non-irradiated chromosomes were less than 5 /im (Fig. 18).

We now can compare diffusion distances with the spot-to-pole distances insuccessful prevention irradiations. The minimum distances between irradiated spotsand poles (Fig. 13D) were greater than 5 /im in 38 % of the 39 cells in which all threesets of autosomal spindle fibres did not disappear; the maximum distances (Fig. 13E)were greater than 5 /ttn in 51 % of the cells, an even larger fraction. Thus, it does notseem likely that the tubulin diffused from the irradiated spot to the pole. Even if oneconsiders 7 /an as the limit of diffusion, based on distances between irradiated spotsand the nearer pole (Fig. 17), the minimum distances were greater than 7 /xm in 20 %of the cells (Fig. 13D) and the maximum distances were greater than 7 /im in 20 % ofthe cells (Fig. 13E). Thus all the evidence suggests strongly that the diffusion fromthe irradiated spot is to the kinetochore, not the poles, and therefore that thekinetochore is the on end of the chromosomal spindle fibre microtubules, not thepole.

Fig. 13. The number of successful prevention irradiations of chromosomes (ordinate)versus the distance between the irradiated spot and the nearer pole (abscissa). (The datado not include one cell in which three .chromosomes were irradiated because theirradiation spot obscured the position of the pole.) A. Distances in 27 cells in which thespindles shortened (see Table 2); the distances plotted are the shorter distances, at theminimum spindle lengths. B. The 12 of the 27 cells in A in which the spindle lengths laterincreased: the distances plotted are the maximum (increased) distances to the nearer pole.C. The 11 cells (for which we have data) in which spindle lengths remained constantduring the irradiation (see Table 2). D. Pooled data from A and C, forming the set ofminimum distances. E. The maximum distances, consisting of those spindles thatshortened and did not elongate again (IS cells from A); the maximum lengths of spindlesthat shortened and then elongated again (i.e. those in B) ; the spindles that did not changelength (i.e. those in c); and the maximum length of the one spindle that lengthenedduring the irradiation without having previously shortened (see Table 2).


8o

Z 3

2

1

0 -r—

A

1

—

B

n n< 0 0 1 2 3 4 5 6 7 8 9 10

Distance (/an)< 0 0 1 2 3 4 5 6 7 8 9 10

Distance (/an)

Z 3

< 0 0 1 2 3 4 5 6 7 8 9 10Distance (/an)

< 0 0 1 2 3 4 5 6 7 8 9 10 > 10Distance (/an)

11

10

98

7

6CJ

4

3

2i

0

1 ,—,— //—E

" I< 0 0 1 2 3 4 5 6 7 8 9 10

Distance (/an)


It is relevant to point out that in all five successful diffusion experiments spindlefibre birefringence remained associated with all three sets of autosomes, i.e. with thetwo farther autosomes as well as the closest one. (There were similar results whenspindle fibres re-formed after colcemid treatment, as described previously (fig. 22 ofCzaban & Forer, 1985).) In two of these five cells the spindle fibres associated withthe chromosomes farthest from the irradiating spot had less birefringence than thoseassociated with the closest chromosome (as judged by eye) but, nonetheless, thebirefringent spindle fibres remained associated with all three chromosomes, evenwhen the farthest chromosome was 7-8/im away from the spot (Fig. 19), that is,farther than the diffusion distance (Fig. 16B).

To summarize, irradiation of chromosomes prevents loss of chromosomal spindlefibre birefringence and this and other data strongly suggest that the kinetochore is theon end of the chromosomal spindle fibre microtubules. Another experimental test ofwhether the chromosomal or polar end of the chromosomal spindle fibre is the on endis to irradiate the polar end directly.

Irradiations of poles. One ideally would like to irradiate both poles simul-taneously ; for technical reasons we were unable to do this and rather irradiated onlyone pole (Fig. 20). The areas of irradiation ranged from 96/im to 406 [im, withenergy fluxes that ranged from l-7Xl(T2/iW to 9-7XlO~2/iW; in only two of thenine irradiations were the irradiating spots within 5 /im of the nearest kinetochore(Fig. 21). Spindle birefringence disappeared in all nine cells in which one pole wasirradiated. If the energy fluxes used in these experiments would have been used toirradiate chromosomes we would have prevented loss of spindle fibre birefringence in

Diffusion experiment

Fig. 14. Diagram indicating areas of cells irradiated in diffusion irradiations. Therectangle, the irradiated spot, is distance dc from chromosome 1, the nearestchromosome; pole A, the nearer pole, is distance dp from the irradiated spot.

Microtubule kinetic polarities in vivo i?

Fig. IS. Successful diffusion irradiation in a cell in a clot preparation. The spindlebecame barrel-shaped but the birefringent chromosomal spindle fibres remained. Thelight rectangle in c is the irradiated spot. The three autosomes are indicated by bars. Theirradiation began at 5*0 min and colcemid was added at 6-0 min; anaphase startedat 55-0 min. A,F,I. Phase-contrast microscopy; B.D.E.G.H, polarization microscopy;C, bright-field microscopy. Times: A, Omin; B, 4-0min; C, 9-0min; D, 18-0min;E, 27-0min; F, 32-0min; G, 42-0min; H, 48-0min; I, 57-0min. X937.

55 % of cells (Fig. 4). Thus, with a 45 % failure rate all nine irradiations would beexpected to be unsuccessful with a frequency of (0-45)9, or 7-6X 10~4. Therefore, weconclude from these data, too, that the kinetochore ends are the on ends of thechromosomal spindle fibre microtubules, not the pole ends.

DISCUSSION

Our results pertain to untreated chromosomal spindle fibre microtubules as theyexist in normal spindles. Our interpretations are based on preventative irradiations:light of wavelength 360 run inactivates colcemid before the colcemid can block the on(assembly) ends of microtubules. Irradiations therefore prevent depolymerization ofchromosomal spindle fibres when the on ends of the chromosomal spindle fibres areirradiated; irradiations do not prevent depolymerization of spindle fibres when theother ends of the chromosomal spindle fibres are irradiated. In our experimentsirradiations that include the kinetochore regions prevent depolymerization of


chromosomal spindle fibres, whereas irradiations of one pole do not. Therefore, thekinetochore ends of the chromosomal spindle fibres are the on ends.

The only one counterargument to this interpretation that we can think of is basedon diffusion of tubulin from the irradiated spot. Tubulin indeed diffuses out of theirradiated spot: irradiations of the cytoplasm (diffusion irradiations) sometimesprevent loss of spindle fibre birefringence, and irradiations of only one or twochromosomes often prevent loss of birefringence from all three autosomal spindlefibres. Thus one might raise the counterargument to our interpretation that in all

0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11

Distance (pm)

100

80

60

40

20

n

\

-A:"'\

\ •\

1571 = 5

>7'n/

B

= 2/T = 3

0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11

Distance (jan)

Fig. 16. A. The percentage of cells in which birefringence remained during diffusionirradiations (ordinate) versus distance from the irradiated spot to the nearest chromo-some (abscissa). B. Data from A pooled with similar data on re-formation of spindle fibresfrom fig. 18c of Czaban & Forer (1985): the percentage of cells with birefringent spindlefibres (ordinate) versus distance from the irradiated spot to the nearest chromosome(abscissa).


0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-1010-11

Distance (/an)> n

Fig. 17. Percentage of cells in which birefringence remained during diffusion irradiations(ordinate) versus distance from the irradiated spot to the nearer pole (abscissa).

successful prevention irradiations free tubulin diffuses to the on ends at the poles.Any interpretation, however, must also explain why the irradiations of the poleregion did not prevent depolymerization of chromosomal spindle fibres; thus onemust extend the counterargument and argue that one can prevent depolymerizationof chromosomal spindle fibres only if both half-spindles are present, i.e. irradiationof both poles is required, one is not sufficient. This certainly is not true in sea-urchinzygotes, however: Hiramoto & Shdji (1982) irradiated one half-spindle (with near-ultraviolet light) as colcemid was added, and the non-irradiated half-spindle lostbirefringence, whereas the irradiated half-spindle did not. Thus they showed thatone half-spindle indeed can remain without the other, that irradiations can indeedprevent depolymerization of one half-spindle only; i.e. therefore they rule out thecounterargument. Nonetheless, it might perhaps be argued that cells of different


species are different in this regard and that one would need to irradiate both poles incrane-fly spermatocytes to prevent loss of spindle birefringence; thus it is worthwhileto look at other aspects of our experiments that also rule out the counterargument.

Diffusion irradiations (of spots in the cytoplasm) sometimes prevented the loss ofchromosomal spindle fibres. The success rate with which the irradiations preventedloss of spindle fibres depended on the distance from the nearest chromosome: thecloser the irradiated spot to the chromosome the more likely that the irradiationwould prevent loss of spindle fibres (Fig. 16). On the other hand, the success rate didnot seem to depend on distance from the nearer pole (Fig. 17). It seems most likely,therefore, that the tubulin diffused to the chromosomes and not the poles, andtherefore that the on ends are at the kinetochores and not the poles.

Another aspect of our data relevant to the counterargument is the prevention fromloss of only some of the chromosomal spindle fibres. If the poles were the on endsthat tubulin in all cases had to diffuse to (in order to prevent loss of birefringence),then there never should be any case in which two sets of spindle fibres remainbirefringent and the third set loses birefringence; i.e. the monomers would reach thepoles and add to the on ends (and prevent loss of birefringence) of all fibres thatterminate at the poles, not just those associated with irradiated kinetochores. Sincethere were two cells in which birefringence remained in only the two spindle fibres

14

12

mbe

r of

cel

ls

» 00

O

3 0Z

4

2

02 3 4 5

Distance (jan)

Fig. 18. Percentage of cells in which birefringence remained associated with all threechromosomes in prevention irradiations of only one or two chromosomes (ordinate)versus distance from the irradiated spot to the non-irradiated chromosome or, in two cellsin which one chromosome was irradiated, to the nearer non-irradiated chromosome(abscissa).

Miavtubule kinetic polarities in vivo 61

0 12 34 56 78 9 10 11Distance to the closest chromosome (/an)

00 1 2 3 4 5 6 7 8 9 10 11

Distance to the furthest chromosome (/an)

0 12 34 56 78 9 10 11Distance to the closest chromosome (fan)

0 1 2 3 4 5 6 7 8 9 10 11Distance to the furthest chromosome (/on)

Fig. 19. The number of chromosomes (ordinate) versus the distance from the irradiatedspot to the chromosome (abscissa) in irradiations in which birefringent chromosomalspindle fibres remained associated with all three autosomes. A. Distance from the nearestchromosome in diffusion irradiations. B. Distance from the farthest chromosome indiffusion irradiations. C. Distance from the nearer non-irradiated chromosome in pre-vention irradiations of one chromosome, D. Distance from the farther non-irradiatedchromosome in prevention irradiations of one chromosome.

associated with the irradiated kinetochores and not the third, and a third cell in whichthe third fibre was of markedly reduced birefringence, the kinetochore ends of themicrotubules must be the on ends, not the pole ends.

A final aspect of our data relevant to the counterargument is the distances fromirradiated spots to poles (when chromosomes were irradiated): these represent thedistances that tubulin would need to diffuse through and not be inactivated bycolcemid were the counterargument to be true. In a large fraction of cases thedistances from the irradiated spots to the nearer poles (Fig. 13) were larger than theempirically determined diffusion distances of tubulin (Figs 16, 17). It does not seempossible for tubulin to diffuse for such distances without being inactivated bycolcemid; therefore the on ends would be at the kinetochores.

We see no way to avoid the conclusion from our data that the on ends ofchromosomal spindle fibre microtubules in normal, untreated spindles are at the


kinetochores. This is the same as the kinetic polarities of chromosomal spindle fibresthat re-form after treatment with colcemid (Czaban & Forer, 1985), and is as

Single pole irradiation

Fig. 20. Diagram of prevention irradiations of single spindle poles.

0 1 2 3 4 5 6 7 8 9 10 11Distance (pan)

Fig. 21. Number of cells (ordinate) versus distance from the irradiated spot to thenearest chromosome in all (unsuccessful) prevention irradiations of poles.


predicted by various authors (e.g. see Margolis, 1978; Margolis, Wilson & Kiefer,1978; Goode, 1981). However, these are opposite to the kinetic polarities predictedby Bergen et al. (1980) and Summers & Kirschner (1979); the data from these lattertwo groups of workers do not rule out alternative interpretations, however, asdiscussed in detail elsewhere (Forer, 1985).

Our conclusion, that the kinetochore ends of chromosomal spindle fibremicrotubules are the on ends, is different from that of Salmon et al. (1984). Theystudied redistribution of fluorescent tubulin after a large region of the spindle (in sea-urchin zygotes) was bleached with a laser and concluded, since the bleached areasseemed to recover uniformly, without movement to or from a pole, that tubulinexchanges with microtubules along the entire lengths of spindle microtubules. Weshould emphasize that our conclusions and experiments pertain to chromosomalspindle fibre microtubules, whereas their experiments were on entire spindle regions,with mixtures of various kinds of spindle microtubules; thus their conclusion, if true,might hold primarily for microtubules other than those of chromosomal fibres andtherefore would not be in disagreement with ours, on chromosomal spindle fibremicrotubules. Further, if different kinds of microtubules were present in the sea-urchin zygote spindles (free microtubules as well as microtubules with differentkinetic polarities) one might have polarized growth but with the impression ofuniform recovery, especially if unbleached microtubules in other focal levels alsoshifted focal levels. Or the absorbed laser energy might have been converted to heatand the local heating could have caused seeding of polymerization. For these andother reasons our conclusions are not necessarily at variance with the data of Salmonetal. (1984).

Our data are relevant to the question of whether or not chromosomal spindlefibre microtubules are continuous from kinetochores to poles in this species (cf.LaFountain, 1976; Fuge, 1984; discussion by Heath, 1981). Our data stronglysuggest that they are, as suggested by LaFountain (1976); otherwise we would not beable to prevent depolymerization by irradiating the kinetochore and would ratherneed to irradiate along the entire lengths of the chromosomal spindle fibres.

Whether a prevention experiment was successful was a function of the energy fluxof the irradiation (Fig. 4). The success in reversing the effect of colcemid and inhaving spindle fibres re-form was also a function of the energy flux of the irradiation(fig. 14 of Czaban & Forer, 1985). However, there may be a difference in the energyfluxes required: re-formation may require a higher energy flux than prevention,because irradiations with energies above 3xlO~2^W were 100% successful inpreventing spindle fibre loss but were not 100 % successful in inducing re-formationof spindle fibres. This difference, if indeed there is one, might be related to theobservation by Amrhein & Filner (1973) that irradiation-induced removal ofcolcemid from tubulin often inactivates the tubulin.

Our irradiations prevented loss of chromosomal spindle fibres but not of astral(and presumably not of interpolar) microtubules. Since anaphase continued none-theless, it is reasonable to suggest that these other microtubules are not necessary formotion but have another role, possibly as a 'glue' to ensure that all the chromosomal


9pindle fibres focus to the poles (discussed by Czaban & Forer, 1985). Be that as itmay, the difference between chromosomal spindle fibre microtubules and the otherspindle microtubules suggests that different concentrations of tubulin might beneeded to maintain the different kinds of spindle microtubules.

In diffusion irradiations of the cytoplasm chromosomal spindle fibres of chromo-somes farther from the irradiated spot did not depolymerize whenever the nearestchromosomal spindle fibres did not depolymerize, even when the fartherchromosomes were beyond the diffusion distance (Fig. 19). Similar complicationsarose in experiments on re-formation of chromosomal spindle fibres after treatmentwith colcemid (Czaban & Forer, 1985). We are puzzled by and cannot explain theseresults, but they may be related to the similarly mysterious "reciprocal kinetochoreinteraction" described by Witt et al. (1980), as discussed previously (Czaban &Forer, 1985).

Portions of this work were presented by B.B.C. to York University in partial fulfilment of therequirements for the M.Sc. degree. The authors acknowledge with gratitude the excellentdesigning and machining of Brian Moore, who greatly facilitated this work. This work wassupported by grants from the Natural Sciences and Engineering Research Council of Canada, theW. Garfield Weaton Foundation (Toronto) and the J. P. Bickell Foundation (Toronto).

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BERGEN, L. G. & BORISY, G. G. (1980). Head-to-tail polymerization of microtubules in vitro.J. Cell Biol. 84, 141-150.

BERGEN, L. G., KURIYAMA, R. & BORISY, G. G. (1980). Polarity of microtubules nucleated bycentrosomes and chromosomes of Chinese hamster ovary cells in vitro. J. Cell Biol. 84, 151-159.

CZABAN, B. B. & FORER, A. (1985). The kinetic polarities of spindle microtubules in vivo, in cranefly spermatocytes. I. Kinetochore microtubules that reform after treatment with colcemid.J. Cell Sci. 79, 1-37.

D E BRABANDER, M. (1982). A model for the microtubule organizing activity of the centrosomesand kinetochores in mammalian cells. Cell Biol. Int. Rep. 6, 901-915.

FARRELL, K. W. & WILSON, L. (1984). Tubulin-colchicine complexes differentially poisonopposite microtubule ends. Biochemistry 23, 3741-3748.

FORER, A. (1985). Does actin produce the force that moves a chromosome to the pole duringanaphase? Can.J. Biochem. Cell Biol. 63, 585-598.

FUGE, H. (1984). The three-dimensional architecture of chromosome fibres in the CTane fly. I.Syntelic autosomes in meiotic metaphase and anaphase I. Chromosoma 90, 323-331.

GOODE, D. (1981). Microtubule turnover as a mechanism of mitosis and its possible evolution.BioSystems 14, 271-287.

HEATH, I. B. (1981). Mitosis through the electron microscope. In Mitosis /Cytokinesis (ed. A. M.Zimmerman & A. Forer), pp. 245-275. New York: Academic Press.

HrRAMOTO, Y. & SHOJI, Y. (1982). Location of the motive force for chromosome movement insand-dollar eggs. In Biological Functions of Microtubules and Related Structures (ed. H. Sakai& G. Borisy), pp. 247-259. New York: Academic Press.

LAFOUNTAIN, J. R. JR (1976). Analysis of birefringence and ultrastructure of spindles in primaryspermatocytes of Nephrotoma suturalis during anaphase. J . Ultrastruct. Res. 54, 333-346.

MARGOLIS, R. L. (1978). A possible microtubule dependent mechanism for mitosis. In CellReproduction: in Honor of Daniel Mazia (ed. E. R. Dirksen, D. M. Prescott & C. F. Fox),pp. 445-456. New York: Academic Press.


MARGOLJS, R. L., WILSON, L. & KIEFER, B. I. (1978). Mitotic mechanism based on intrinsicmicrotubule behaviour. Nature, Land. 272, 450-452.

MclNTOSH, J. R. (1984). Mechanisms of mitosis. Trends Biochem. Set. 9, 195-198.MCINTOSH, J. R. & EUTENEUER, U. (1984). Tubulin hooks as probes for microtubule polarity: an

analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle.J.CellBiol. 98, 525-533.

PICKETT-HEAPS, J. D. & TIPPIT, D. H. (1978). The diatom spindle in perspective. Cell 14,455-467.

PICKETT-HEAPS, J. D., TIPPIT, D. H. & PORTER, K. R. (1982). Rethinking mitosis. Cell 29,729-744.

RlEDER, C. L. (1982). The formation, structure, and composition of the mammalian kinetochoreand kinetochore fiber. Int. Rev. Cytol. 79, 1-58.

SALMON, E. D., LESLIE, R. J., SAXTON, W. M., KAROW, M. L. & MCINTOSH, J. R. (1984).

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeledtubulin and measurements of fluorescence redistribution after laser photobleaching. J. Cell Biol.99, 2165-2174.

SUMMERS, K. & KIRSCHNER, M. W. (1979). Characteristics of the polar assembly and disassemblyof microtubules observed in vitro by darkfield light microscopy. J. Cell Biol. 83, 205-217.

TELZER, B. R. & HAIMO, L. T. (1981). Decoration of spindle microtubules with dynein: evidencefor uniform polarity. J . Cell Biol. 89, 373-378.

TIPPIT, D. H., PICKETT-HEAPS, J. D. & LESLIE, R. (1980). Cell division in two large pennatediatoms HantzscMa and Nitzschia. III . A new proposal for kinetochore function duringprometaphase.J. Cell Biol. 86, 402-416.

WITT, P. L., Ris, H. & BORISY, G. G. (1980). Origin of kinetochore microtubules in chinesehamster ovary cells. Chromosoma 81, 483-505.

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{Received 25 February 1985 -Accepted 26 July 1985)

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THE KINETIC POLARITIES OF SPINDLE …jcs.biologists.org/content/joces/79/1/39.full.pdf · (assembly) ends of treadmilling microtubules (Farrell & Wilson, 1984), ... chromosomal spindle