an estimate of the amount of microtubule protein …procedure was the same as in (a), except that...

J. Cell Sci. 6, i59~'76 ("97°) 159Printed in Great Britain

AN ESTIMATE OF THE AMOUNT OF

MICROTUBULE PROTEIN IN THE ISOLATED

MITOTIC APPARATUS

W. D. COHEN* AND L. I. REBHUNDepartment of Biology, Princeton University, Princeton, New Jersey, U.S.A.

SUMMARY

The microtubule content of the isolated mitotic apparatus of sea-urchin eggs (Arbaciapunctulata) has been investigated by electron microscopy. Cross-sections were made throughasters or spindles of flat-embedded mitotic apparatuses of known mitotic stage and specificorientation in the block. Cross-sections between chromosomes and poles of five metaphasehalf-spindles revealed approximately 2000-2300 sectioned microtubules. The number wassomewhat higher in three anaphase half-spindles examined, approximately 2400-2600.

A method was devised for calculating the total number of microtubules in an aster, basedupon the number of microtubules appearing in cross-sections. Application of this method toselected mitotic apparatuses enabled calculation of the total number of microtubules in meta-phase mitotic apparatuses of average dimensions. Using a 13-protofilament model of the micro-tubule and existing data on possible monomer sizes and molecular weights, the total amount ofmicrotubule protein in the isolated mitotic apparatus was calculated. The values obtained are inthe range of about 1—2 x io~8 mg microtubule protein per isolated mitotic apparatus. Thesevalues are close to those reported for the 4-5 S protein of the isolated mitotic apparatus, but areconsiderably lower than the amount of 22s protein. The results are discussed with respect tocellular factors which determine microtubule number, and the possible sources and origin ofmitotic microtubule protein.

INTRODUCTION

The isolated mitotic apparatus of marine eggs has been the object of considerablestudy, at both the ultrastructural and biochemical levels. The ultrastructural com-ponent of the mitotic apparatus which is currently of major interest is the mitoticmicrotubule, a structure suspected of playing a skeletal and perhaps motile role inmitotic movements. The isolated mitotic apparatus is, potentially, an excellent sourceof microtubules and of microtubular subunits. There have been several studies of thestructure of mitotic microtubules, as well as of those components, present in extracts ofthe isolated mitotic apparatus, which might be the molecular subunits of which themicrotubules are constructed (Barnicot, 1966; Sakai, 1966; Kiefer, Sakai, Solari &Mazia, 1966; Kane, 1967; Stephens, 1967). Identification of the latter is of increasedimportance because of the general structural similarity between mitotic microtubulesand those found in other systems, such as cilia and flagella; comparison of microtubularsubunits from different sources could provide much information about microtubulefunction. In addition to such structural problems, the mitotic microtubule protein of

Present address: Department of Biological Sciences, Hunter College of the City Universityof New York, New York, N.Y., U.S.A.

160 W. D. Cohen and L. I. Rebhun

marine eggs deserves scrutiny because of the question as to whether it is synthesizedin vivo during early cleavage.

In studies such as these, a useful piece of information would be a value for theamount of microtubule protein in the mitotic apparatus. The purpose of this paper isto provide an estimate for this quantity using morphological procedures.

MATERIALS AND METHODS

The isolated mitotic apparatus of the egg of the sea-urchin Arbacia punctulata was the objectof study. Gametes were obtained by the electrical method (Harvey, 1956). Fertilization mem-branes were removed using thioethylgluconamide (Mazia, Mitchison, Medina & Harris, 1961)as follows: diluted sperm was added to a suspension of washed eggs in artificial sea water(Harvey, 1956) at 20-22 °C. After stirring for 30 s, an equal volume of calcium-free sea watercontaining thioethylgluconamide at 2 mg/ml (pH about 8) was added and stirring continued forabout 10 min. The eggs were then passed through fine silk mesh, and, after settling, were re-suspended in calcium-free sea water. Development proceeded at 20-22 °C.

The mitotic apparatus was isolated just prior to first cleavage, according to the method ofKane (1962, 1965), and was stabilized after isolation by exposure to Mg2+ (Goldman & Rebhun,1969). Variations in details of procedure used for different preparations of mitotic apparatus areas follows:

(a) The isolation medium was 12 % hexylene glycol, 001 M maleate, pH 62 . Eggs were lysedin the medium by brief swirling on a vortex mixer. The preparation was placed in a bath at o °C,and one-tenth volume of 0011 M MgCl2 in isolation medium added, to give a final concentrationof 0001 M MgCl2. The mitotic apparatuses were washed 5 times in isolation medium containing0001 M MgClj, at 0-4 °C, and resuspended in the same medium.

(b) The isolation medium was 12 % hexylene glycol, 001 M phosphate, pH 64 . Procedure wasthe same as in (a), except that MgCl. was added to 0-003 M, and the mitotic apparatuses washed5 times in isolation medium containing 0-003 M MgCl... Some of the spindles lost their astersduring these washes.

(c) Procedure was the same as in (b), except that the 5 washes were omitted; mitotic appara-tuses were sedimented once and resuspended in isolation medium containing MgCl..

Immediately after preparation, mitotic apparatuses were fixed. Final resuspension in isolationmedium containing Mg1+ was followed by addition of an equal volume of the same mediumcontaining 5 % glutaraldehyde (pH adjusted to that of isolation medium). The mitotic appara-tuses were fixed at o °C for 60 min, sedimented, washed once with water, and post-fixed in1 % OsO.i in water for 45-60 min. After passage through an ethanol dehydration series andpropylene oxide, they were flat-embedded in Epon 812 (Luft, 1961), using Beem capsule capsor carbon-coated plastic dishes as moulds.

Individual embedded mitotic apparatuses were observed under phase contrast, and trimmingwas carried out so that a particular pre-selected mitotic apparatus was oriented with its long axisapproximately perpendicular to the face of the block. Sections (cross-sections) were cut on aSorvall MT-2 ultramicrotome, picked up on Formvar- and carbon-coated grids, stained withuranyl acetate (Stempak & Ward, 1964) followed by lead citrate (Reynolds, 1963), and examinedin a Hitachi HS-7S electron microscope. The entire mitotic apparatus was cross-sectioned,beginning at the outer edge of one aster. When approximately 50 sections (non-serial) had ac-cumulated, they were picked up on grid no. 1, the next 50 on grid no. 2, and so on. The gridswere stored in sequence, each representing a sectioned length of about 4 to 5 /im. Thus, theapproximate location of a section within a mitotic apparatus was indicated by its grid number.

The phase-contrast microscope used was a Zeiss Winkel instrument with which an ocularmicrometer reticule was employed for measurements of embedded mitotic apparatuses. Someobservations were also made with a Zeiss photomicroscope equipped with Nomarski differentialinterference optics or polarizing optics.

Counts were made of all of the microtubules found in an entire cross-section through spindleor asters. The majority of microtubules appeared in cross-section, and were rather easilycounted (Figs. 7, 8). At the periphery of the mitotic apparatus, especially in the asters, micro-

Microtubule content of mitotic apparatus 161

tubules were sectioned obliquely, as expected. Some of these proved more difficult to identify asmicrotubules, especially ones which were surrounded by dense material. Questionable micro-tubules did not amount to more than about 5 or 6%, however, and for consistency they werealways counted.

RESULTS

Initial counts of the number of microtubules in isolated spindles

In the initial phase of this work, counts were made of the number of microtubulesobserved in cross-sections between chromosomes and poles of 6 different spindles.These are presented in Table 1. Both metaphase and anaphase spindles from several

Table 1. Numbers of microtubules observed in cross-sections betweenchromosomes and poles of several isolated spindles

No. of micro-Preparation tubules counted

Spindle no. Mitotic stage procedure* in half-spindle

I

*t3t456

MetaphaseMetaphaseMetaphaseAnaphaseAnaphaseAnaphase

abbbaa

196421602314239324752610

• The letters refer to the letters denoting variations of isolation procedure as given underMaterials and Methods.

f Spindles 2 and 3 were aster-less; the others were spindles of intact mitotic apparatuses.

different preparations of mitotic apparatuses were examined. There were slightvariations of details in different preparations, as described under Materials andMethods. As seen in Table 1, the number of microtubules in the 3 metaphase half-spindles examined ranged from approximately 2000 to 2300, while the 3 anaphasehalf-spindles exhibited a somewhat greater number, approximately 2400 to 2600.

The total number of microtubules in ' the' isolated mitotic apparatus

In any preparation isolated mitotic apparatuses vary in size. Those in anaphase tendto be larger than those in metaphase, for example, as observed by Mazia (1955).However, variation in size may also be observed amongst those in the same mitoticstage. Since there might be a relationship between the size of the isolated mitoticapparatus and its microtubule content, this variable must be taken into considerationwhen attempting to determine 'the' number of microtubules in 'the' isolated mitoticapparatus.

For the purposes of this work, two approaches were possible: (i) a large sample ofmitotic apparatuses could be examined, and the average number of microtubules de-termined, or (ii) the average size of the mitotic apparatus after fixing and embedding

II C EL 6


could be determined, and then the number of microtubules determined for a mitoticapparatus of average size. The latter approach was adopted.

The average size of the mitotic apparatus after isolation, fixation, and embedding

To limit variables, mitotic apparatuses at the same mitotic stage—metaphase—were employed. This was the most prevalent stage in our preparations. Measurementswere made on ioo metaphase mitotic apparatuses after fixation and embedding. The

70

«j 60

I 50

2 40

£30

S.20

10 II 1I

EstimatedI . ., I

spindle

<-22%< n

Mitotic apparatusI — 1

length

I I I A20- 22- 24- 26- 28-21 23 25 27 29

Estimated spindlelength0'm)

42- 44- 46- 48- 50- 52- 54- 56- 58- 60 -43 45 47 49 51 S3 55 57 59 61

Mitotic apparatus length

18- 2 0 - 22- 24 - 26- 28 - 30 -19 21 23 25 27 29 31

Aster diameter(jtm)

Fig. i. Measurements of ioo metaphase mitotic apparatuses after fixation, dehydra-tion, and flat embedding in Epon.

Table 2. Measurements made on 100 metaphase mitotic apparatuses after fiatembedding in Epon {preparation procedure (c); see Materials and Methods)

Measurement made

Average measure-ment (jim) ( = total

length/100)

Measurement classwith greatest

frequency (jim)

Mitotic apparatus lengthAster diameterSpindle length (estimated)

2524

24-2524-25

criterion for choice of a particular mitotic apparatus for measurement was only that itwas clearly at metaphase. Figure 1 presents these measurements, consisting of thelength of the mitotic apparatus from the edge of one aster to the edge of the other, theaster diameter, and the estimated length of the spindle from pole to pole (position of


spindle poles taken as the apparent projected convergence point of spindle fibres; referto diagram in Fig. 1). The data are summarized in Table 2. The average dimensionsdescribe a mitotic apparatus consisting of two spheres, side-by-side, each with a radiusof approximately 12-5 /tm, with a spindle approximately 25 /tm (2 radii) long (Fig. 2).This also describes the mitotic apparatus most frequently encountered in the prepara-tion (Table 2, end column).

Definition of' one microtubule'

The total number of microtubules in one mitotic apparatus is the number in thespindle plus the number in the asters. A determination of this number requires precisedefinition of what is meant by one microtubule. Considering the dimensions of theaverage mitotic apparatus described above, the most convenient definition for the

-One astral andone spindle microtubule

Metaphase'plate'

5CV m

Fig. 2. The embedded metaphase mitotic apparatus of average dimensions, and thedefinition of 'one microtubule' with respect to these dimensions.

purposes of this paper is as follows: 'one microtubule' is a microtubule 12-5/tm(= one radius) in length. Thus, a microtubule running from the centre of the aster toits periphery is one microtubule, and a microtubule running from spindle pole tometaphase plate is one microtubule (Fig. 2). A continuous microtubule running frompole to pole would therefore count as two microtubules.

Method for calculation of the number of microtubules in an aster

The method employed for the calculation of the number of microtubules in an asterwas based upon the following assumption: the microtubules of the aster radiate fromthe centre of the aster with approximately spherical symmetry (except for the half-spindle volume; see correction below). The number of microtubules within the astralsphere (= iVa_g_) can then be calculated from the number observed passing through across-section taken between the centre of the sphere and its periphery, as in Fig. 3.Such a section will be circular, and this circle will be both the base of a cone (whoseapex is at the centre of the sphere) and the base of a spherical segment adjacent to thecone (Fig. 4). The total volume of the cone plus the spherical segment is equal to§nr2h, where h is the distance from the surface of the sphere to the centre of the base(Lines, 1965). The number of microtubules radiating from the apex and passing


through the circular section is therefore the number occupying the volume \irr2h. Theratio of this volume to the total volume of the sphere is

Thus, if the volume occupied by the half-spindle is included temporarily (correctionbelow), then the total number of microtubules in the aster will be the number passingthrough the circular section multiplied by ir\h. The value of h is easily determined.Referring to Fig. 5, r is known ( = 125 fim) and a is known ( = one-half the diameterof the circular section). Therefore, b can be calculated (a2 + b2 = r2). The value of h isthen r —b.

Microtubules radiating within cone

Volume occupied byhalf-spindle

• Plane of crosi-section

Fig 3. Side view of cross-section through astral sphere.

Sectioned microtubules

Sphericalsegment Cone

Circular section =base of cone and ofspherical segment

Fig. 4. Geometrical basis for calculation of number of microtubules per aster.

Correction of astral sphere volume for volume occupied by the half-spindle

In the calculation of the total number of microtubules in one aster (Na_s_ above) the'aster' is taken to be the entire sphere. However, as is apparent in Fig. 3, part of thevolume of this sphere is occupied by the conical half-spindle inserted into it. Theassumption of uniform radial density of astral microtubules applies only to the non-spindle portion of the astral volume, for within the half-spindle volume the radial


density of microtubules is considerably greater than elsewhere. Therefore, the 'truevolume' of the aster is the total volume of the astral sphere minus the volume of thehalf-spindle, and it is only the microtubules in this true volume which must be countedas astral microtubules.

The volume of the half-spindle can be approximately determined by consideringthe metaphase plate as the equivalent of a section passing through the aster. Thevolume of the half-spindle (^/2) will then be f (nr2hM/2), and, following the calculationmade in the previous section, the fraction of total astral sphere volume which the half-spindle occupies will be (AN/2)/2r. The value of h^^ is determined as described in theprevious section for h\ however, in this case the value for length a (refer to Fig. 5) is

Fig. 5. Lengths used in calculation of number of microtubules per aster. The volume ofthe spherical segment plus the volume of the cone = 2/3 TTr%h, where h — distancefrom surface of sphere to centre of base.

Table 3. Data and calculated fractions used in correction of number of microtubulesin the astral sphere (Na 8 ) for those in the volume occupied by the half-spindle

Approx.measuredspindle

diameter (/4m)

Fraction of astralsphere volume Fraction of astral

calculatedoccupied byhalf-spindle

sphere micro-tubules in the

true aster

u-5 S'6/ioo 94-4/100

now one-half the diameter of the metaphase plate. The diameter of the latter is ob-tained by measurement of embedded spindles of the average-sized mitotic apparatuses.

Of the total number of microtubules in the astral sphere (N& a), those in the volumefraction (As/2)/2r are to be excluded. Multiplication of Nag by [1 — (As/2/2r)] will thusgive the corrected number of microtubules in the true aster (Na).

The data and calculated fractions for correction of N& 8 are presented in Table 3.

The total number of microtubules in the isolated mitotic apparatus

The total number of microtubules in the isolated mitotic apparatus, based on thedefinition of one microtubule given above, will be equal to the number in the two

i66 W. D. Cohen and L. I. Rebhun

asters (corrected for spindle volume) plus the number in the two half-spindles. For thepurpose of calculation, it is assumed that the number in one aster is the same asthe number in the other aster, and similarly, that the number in one half-spindle is the

Table 4. The total number of microtubules in the true aster (Na)

Mitoticapparatus

no.*

I

II

Astralsection

no.f

1

2

3

1

Sectiondiameter

w142 0

14

22

T

Own)

1 2 5

12-512-5

12-S

Calcu-lated h

(jim)

2 - 2

5 O

2 - 2

6-6

No. of micro-tubules coun-

ted in sec-tion (N)

1152 6 0

138

339

Calculated no.of microtubu-les in astral

sphere (JV,, =N x 2r/h)

13001300

157°1288

Corrected no. ofmicrotubulesin true aster

(N, = 0-944 x

1227 j1227 average1482

1216

= 1312

• Preparation procedure c; see Materials and Methods.f Sections through the asters of mitotic apparatuses I and II were located approximately as follows:

Mitotic apparatus

Mitotic apparatus II

Table 5. The total number of microtubules in the mitotic apparatus

Total no. micro-Mitotic No. microtubules No. microtubules tubules in mitotic

apparatus in half-spindle in one aster apparatusno.* (a) (b) (20 + 26)

I 2°S4 1312(av. of the 3

sections)

1216

6732

II 2128 1216 6688

• Preparation procedure c; see Material and Methods.

same as in the other half-spindle. Data and calculation of the total number of micro-tubules in two metaphase mitotic apparatuses of average size is presented in Tables4 and 5. Table 4 gives the total number of microtubules in the aster (JVa; corrected forspindle volume), while Table 5 gives the total number for the entire mitotic apparatus.


For the two mitotic apparatuses in Table 5 (I and II) the total numbers were 6732 and6688, respectively, or approximately 6700 microtubules per mitotic apparatus.

Figure 9 shows mitotic apparatus I prior to sectioning, and the appearance of cross-sections through aster and spindle are illustrated in Figs. 10 and 11, respectively.

DISCUSSION

Possible sources of error

Among possible sources of error which have been considered are the problems ofmicrotubule stabilization after isolation of the mitotic apparatus, and microtubulepreservation by fixation. Thus Mg2+ was used in the medium subsequent to isolation,following the demonstration by Goldman & Rebhun (1969) that Mg2+ will preventdecrease of birefringence and loss of microtubules during washes in isolation medium.Moreover, the fixatives employed are known to preserve the birefringence of the iso-lated mitotic apparatus (Goldman & Rebhun, 1969; Rebhun & Sander, 1967), and wehave confirmed this with the preparations employed in this work. Since most of thebirefringence appears to be produced by the mitotic microtubules (Rebhun & Sander,1967), the fixatives probably preserve most of them.

It is unlikely that significant numbers of microtubules were lost from the interiorof the isolated mitotic apparatus by mechanical disruption during isolation. The num-ber of microtubules counted in metaphase mitotic spindles which had had their astersknocked off mechanically (Table 1, spindles 2 and 3) was about the same as the numberin spindles of intact mitotic apparatuses (Table 1, spindle 1; Table 5, spindles ofmitotic apparatuses I and II). All were in the range of about 2000-2300 per half-spindle. Furthermore, the total number of microtubules in the metaphase mitoticapparatus of average size was determined for mitotic apparatuses which had not beensubjected to the stress of repeated centrifugation and resuspension (see Materials andMethods, preparation (c)).

Possible errors in the counting may also be considered. The great majority ofmicrotubules were readily identifiable in both transverse or oblique section. Asmall percentage (5—6%) which were questionable were counted as microtubules, soas to regularize the counts. While most of the microtubules appearing in the cross-sections through the spindle were spindle microtubules, some at the periphery,generally appearing in oblique section, were probably those of the aster. Howeverthey could not be definitely identified as such, and were therefore counted as spindlemicrotubules. Some error is also associated with the assignment of a microtubulelength of 12-5 /tm. Most of the astral microtubules are probably not as long as 12-5 /imsince it is observed that they usually do not penetrate all the way to the centre of theaster. Thus, the use of the figure 12-5 fim per microtubule tends to maximize the totallength of microtubules, calculated below.


The total amount of microtubule protein in an isolated metaphase mitotic apparatus ofaverage size

The following data may be used to estimate the total amount of protein contained inthe microtubules of one metaphase mitotic apparatus of average size:

(a) There are approximately 6700 microtubules in the isolated mitotic apparatus,each 12-5 fim long (Table 5).

(b) There are 13 protofilaments per microtubule (Ledbetter & Porter, 1963, 1964;Kiefer et al. 1966).

(c) Each protofilament consists of a series of approximately spherical protein mono-mers which are about 50 A in diameter (Grimstone & Klug, 1966) and have a mole-cular weight of about 55 000 (Renaud, Rowe & Gibbons, 1968). (These figures are usedassuming that the microtubules of the mitotic apparatus and the outer microtubules ofcilia have similar subunits; a different set of data is used in another calculation below.)

The total length of protofilament in one mitotic apparatus will be

6700 x 13 x 12-5 /im = 1088750 /tm.

If this length is constituted by monomers 50 A in diameter the total number of mono-mers will be 1 088750 x io*/5O, which is approximately 1-09 x io10/5o, giving 2-18 x ioR

monomers per mitotic apparatus. Since 6-02 xio23 monomers = 55000 g, 2-18 xio8

monomers = 1-99 x io~8 mg.If, instead of a molecular weight of 55000 and a diameter of 50 A, one assumes a

molecular weight of 34000 and a diameter of 35 A (Sakai, 1966; Kiefer et al. 1966;data for protein believed to be subunits of mitotic microtubules), the number ofmonomers will be 1088750 xio4/35 = 3-11 xio8 per mitotic apparatus, and theamount of microtubule protein will then be 1-76 x io"8 mg.

Another calculation may be made by assuming that the microtubule protein has adensity of 1-35 g/cc. If each monomer is a sphere 50 A in diameter, then the volumeof one monomer will be 6-53 x io^/tm3. The total volume of monomers= 2-18 x io8 x6-53 x io"8 /tm3 = 14-23/tm3. If the density is 1-35 x 1 o~9 mg//tm3

(1-35 g/cc), then the calculated total volume of monomers represents 1-92 xio~8mgmicrotubule protein per mitotic apparatus. For a spherical monomer 35 A in diameter,the volume of one monomer will be 2-25 x io~8/tm3 and the total volume of mono-mers is 3-11 xio8 X2-25 xio~8/<m3 = 7-00/tm3, representing 0-95 xio~8mg micro-tubule protein per mitotic apparatus.

Comparison of calculated amount of microtubule protein per mitotic apparatus with amountof'' 22 s' protein per mitotic apparatus

According to Kane (1967), one isolated mitotic apparatus contains 16-0 x io~8 mgprotein. Approximately one-half of this or 8-o x io"8 mg is 22s protein. However, ascalculated above, the microtubules can contribute at most about 2-0 x io~8 mg protein,so that there is at least 4 times as much 22 s protein as can be accounted for by micro-tubules. It is unlikely that the amount of microtubule protein calculated above is toolow; wherever a choice was possible, the data were maximized. It is also unlikely that


the amount of 22 s protein calculated by Kane (1967) is too high, for he reports that thevalue may in fact be too low because it is based upon 100% synchrony, yield, andrecovery of mitotic apparatuses in a preparation. Therefore, either the 22 s proteindoes not come from the mitotic microtubules, or it comes from both the mitoticmicrotubules and from some other site within the isolated mitotic apparatus. Theformer is, of course, the simpler possibility. Renaud et al. (1968) have already suggestedthat 22 s protein may come from a mitotic apparatus matrix rather than the micro-tubules, on the basis of lack of similarity to the outer fibre protein of cilia. Stephens(1968) has suggested that the 22 s protein is not a subunit or precursor of the micro-tubule, based on lack of similarity of its minimal subunit weight and amino-acidcomposition to that of protein from the outer fibres of sea-urchin flagella. In addition,Borisy & Taylor (1967) have demonstrated that the 22 s protein does not bind col-chicine to the extent expected of microtubule protein.

If the 22 s material is not microtubule protein, then what protein is? As reported byKane (1967) two peaks appear upon ultracentrifugation of the fraction soluble inO-6M KC1 obtained from the glycol-isolated mitotic apparatus: one, the 22s material,(constituting about 80% of the protein of the fraction) and the other, material sedi-menting at 4-5 s. If this 4-5 s material constitutes the remaining 20% of the KC1-soluble fraction, then it is present in about one-fourth the amount of the 22 s protein,or approximately 2-0 x io~8 mg per mitotic apparatus. This figure is in striking agree-ment with the amount calculated above on the basis of a monomer diameter of 50 Aand a molecular weight of 55000. Moreover, our calculated range of values for thepercentage of mitotic apparatus protein contributed by microtubules (about 6-12%,assuming the total protein per mitotic apparatus to be 16-0 x io~8 mg) is in reasonableaccord with the amount of colchicine-binding protein present in mitotic apparatusextracts (Borisy & Taylor, 1967).

Although the material which sediments at 4-5 s was described as heterogeneous byKane (1967), the spread of sedimentation values given is not great, and may originatefrom partial denaturation of the microtubule protein during the isolation or extractionof the mitotic apparatus. Recent determinations of the amino-acid composition andmolecular weight of the 4-5 S component have shown that it resembles microtubuleprotein from sea-urchin sperm flagella (Stephens, 1968), lending support to its tenta-tive designation as dissolved mitotic microtubule protein.

If our estimates are approximately correct, then microtubule protein can account foronly one-eighth of the total protein of the isolated Arbaciapunctulata mitotic apparatus.The question then arises as to where the remaining seven-eighths come from. We canonly state the possibilities: ribosomes, membranes of vesicles, chromosomes, and apostulated 'matrix' or gell-like component. The existence of the latter is suggested bythe polyelectrolyte behaviour of the isolated mitotic apparatus (Cohen, 1968).

The number of microtubules in the spindle

There are many questions which can be framed concerning the number of micro-tubules in the mitotic (or meiotic) spindle. It is apparent that the number of micro-tubules in the spindles of cells of different species, or even of different types of cells of


the same species, will not be the same. There is also the possibility that the numbermight vary with mitotic stage, and it is of interest that the 3 anaphase spindles examined(Table 1, spindles 4-6) all had a somewhat greater number of microtubules than the5 metaphase spindles (Table 1, spindles 1-3; Table 5, spindles of mitotic apparatusesI and II). However, there are as yet insufficient data for these differences to be con-sidered significant.

Which variables might be expected to determine microtubule number in a normalcell undergoing mitosis in its normal environment? Which parts of the cell might bedirectly involved? Is the number of microtubules a function of the number of chromo-somes present, or of their size? Do chromosomes have a genetically determinednumber of sites to which microtubules can attach at the kinetochore? Is the number ofcontinuous microtubules dependent upon the same controlling factors as chromosomalmicrotubules? There are relatively few data which can be brought to bear on thesequestions at present. A few preliminary observations are perhaps worthwhile, however.It appears unlikely that the number of chromosomes alone bears a primary relationshipto the total number of microtubules. This is illustrated by the fact that crane-flyspermatocytes, which have only 5 chromosomes and chromosomal fibres per half-spindle, contain about 1700 microtubules in the half-spindle (Behnke & Forer, 1966).A comparable number of microtubules, about 2000-2300, is found in the isolatedhalf-spindle of A.punctulata (Tables 1 and 5), a species which has 36 or perhaps 38 chro-mosomes per half-spindle (Harvey, 1956). It is more likely,that there will be greatvariation in the number of microtubules attaching at the kinetochore of differentchromosomes in different species. It has been estimated that in Haemanthus there are50-100 microtubules per metaphase kinetochore (Harris & Bajer, 1965), while 4-7have been found per kinetochore in cells of foetal rats (Jokelainen, 1967), and initialestimates for the isolated A. punctulata spindle suggest 10-20 (W. D. Cohen, un-published observations). A primary determinant of microtubule number may thus bethe number of sites per kinetochore, with the number of kinetochores secondary.

The origin of the protein constituting the microtubules of the isolated mitotic apparatus

The high value calculated for microtubule protein content is about 2-0 xio"8 mg.Within the probable range of values of total protein content per egg of A. punctulata,the lowest value is 3-0 x io~6 mg protein per egg (Kane, 1967). Using these figures, themaximum percentage of total egg protein constituting the microtubules of the isolatedmitotic apparatus is about 0-067%. The question may be raised as to whether such anamount of protein could be synthesized in the time interval between fertilization andmetaphase, or whether it must have been present in the unfertilized egg. Wilt, Sakai &Mazia (1967) estimate that the amount of protein synthesized between fertilization andfirst cleavage in S. purpuratus eggs may be as high as 0-5 % of the total egg protein, andGross & Cousineau (1963) calculated at least 0-17% for A. punctulata. The latterfigure is more than twice as great as the calculated percentage of microtubule protein.Thus, considering cellular synthetic capacity only, it must be concluded that all of themicrotubule protein found in the isolated mitotic apparatus could be synthesizedsubsequent to fertilization.


However, extension of this conclusion to encompass all microtubule protein presentin the mitotic cell in vivo is unwarranted. The isolated mitotic apparatus may notcontain all of the mitotic microtubules in the cell. Outer portions of astral micro-tubules may be left behind in the egg lysate during isolation of the mitotic appara-tuses. As a rough basis for estimation of such loss, it can be assumed that the astralrays of the mitotic apparatus in vivo extend from the centre of the aster to the peri-phery (cortex) of the cell (as discussed by Dan, 1943). If the egg is 75 /tm in diameter(Harvey, 1956) and the isolated mitotic apparatus 50 //m in length, then each astralmicrotubule would have to be extended by a minimum of 12-5 fim in order to reach the

12 5 , 12 5 1 12 5 , 12 5 , 12 5 , 12 5 .

75Fig. 6. Basis for estimating the number of mitotic microtubules in situ.

Measurements in micrometres.

periphery (Fig. 6). Based on a model of an aster containing 1300 microtubules of uni-form radial density (Table 4), each 12-5 /tm long, all of the astral microtubules wouldhave to be extended by an average of about 21-5 /tm to reach the periphery. Assumingthis 21-5-/4111 portion to be left behind during isolation, mitotic apparatuses I and II(Table 5) would then contain, in vivo, the equivalent of 11200 and 10840 microtubulesrespectively (each one 12-5 /tm long). Using a figure of 11 200, the microtubule proteincontent would be 3-35 xio~8 mg, corresponding to o-n% of the total egg protein.This is still less than the amount of protein estimated to be synthesized betweenfertilization and first cleavage (0-17% of total egg protein, or higher; Gross &Cousineau, 1963).

There are, however, two additional factors which must be considered. The first isthat, prior to first cleavage, sea-urchin eggs probably contain much more microtubuleprotein than that which is actually incorporated into the structure of the normal mitoticapparatus in vivo. This is indicated by D2O and hexylene-glycol treatment of marineeggs, which markedly increases the size and birefringence of the mitotic apparatus invivo(Rebhun, 1966; Inoue & Sato, 1967). Although evidence for a corresponding increase inmicrotubule number has not yet been published, the general correlation between bire-fringence and microtubules makes such an increase likely. The second factor concerns


the nature of the protein synthesized subsequent to fertilization. As described by Gross

(1967), this protein consists of many different molecular species. Thus, of the total

amount of new protein, only a small fraction could consist of any one molecular species

such as microtubule protein. Considering this, plus the observation that the cell

normally may contain excess mitotic microtubule protein, it is unlikely that the syn-

thesis of all of the microtubule protein takes place after fertilization.

Finally, it is of interest to consider whether the protein constituting the micro-

tubules of the flagellum of the fertilizing spermatozoon could make a significant con-

tribution to formation of the mitotic microtubules. The tail of the Arbacia punctulata

sperm is approximately 45 /tm long (Harvey, 1956), and contains 9 peripheral doublets

and 1 central microtubule pair. There are thus 20 microtubules running approximately

45 /jm for a total microtubule length (single) of 900 /tm and a protofilament length of

about 13 X900 = 11700/tm. This is only slightly more than 1% of the calculated

length of protofilament in one isolated mitotic apparatus (more than io6 /tm), showing

that the potential contribution from this source is negligible.

This investigation was supported, in part, by Public Health Service Postdoctoral fellowshipno 1-F2-CA-33, 181-01 to Dr W. D. Cohen. Additional support was provided by grants toDr L. I. Rebhun from The National Science Foundation and The National Institutes of Health.

REFERENCES

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crane-fly (Nephrotoma sutaralis Loew): Intranuclear and intrachromosomal microtubules.C. r. Trav. Lab. Carlsberg 35, 437~455'

BORISY, G. & TAYLOR, E. W. (1967). The mechanism of action of colchicine. J. Cell Biol. 34,535-543-

COHEN, W. D. (1968). Polyelectrolyte properties of the isolated mitotic apparatus. Expl CellRes. 51, 221-236.

DAN, K. (1943). Behavior of the cell surface during cleavage VI. On the mechanism of celldivision. J. Fac. Sci. Tokyo Univ. 6, 323-368.

GOLDMAN, R. D. & REBHUN, L. I. (1969). The structure and some properties of the isolatedmitotic apparatus. J. Cell Sci. 4, 179—209.

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GROSS, P. R. (1967). The control of protein synthesis in embryonic development and differen-tiation. In Current Topics in Developmental Biology, vol. 2 (ed. A. A. Moscona & A. Monroy),pp. 23-26. New York and London: Academic Press.

GROSS, P. R. & COUSINEAU, G. H. (1963). Synthesis of spindle-associated proteins in earlycleavage. J. Cell Biol. 19, 260-265.

HARRIS, P. & BAJER, A. (1965). Fine structure studies on mitosis in endosperm metaphase ofHaemanthus katherinae Bak. Chromosoma 16, 624-636.

HARVEY, E. B. (1956). Tlie American Arbacia and Otlter Sea Urchins. Princeton: PrincetonUniversity Press.

INOUE, S. & SATO, H. (1967). Cell motility by labile association of molecules. J. gen. Physiol.50 (no. 6, part 2), 259-288.

JOKELAINEN, P. T. (1967). The ultrastructure and spatial organization of the metaphasekinetochore in mitotic rat cells. J. Ultrastntct. Res. 19, 19-44.

KANE, R. E. (1962). The mitotic apparatus: isolation by controlled pH. J. Cell Biol. 12, 47-55.KANE, R. E. (1965). The mitotic apparatus. Physical-chemical factors controlling stability.

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KANE, R. E. (1967). The mitotic apparatus. Identification of the major soluble component of theglycol-isolated mitotic apparatus. J. Cell Biol. 32, 243-253.

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LINES, L. (1965). Solid Geometry. New York: Dover Publications.LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem.

Cytol. 9, 409-414.MAZIA, D. (1955). The organization of the mitotic apparatus. Symp. Soc. exp. Biol. 9, 335-357.MAZIA, D., MITCHISON, J. M., MEDINA, H. & HARRIS, P.( i96i) .The direct isolation of the mito-

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(Received 6 February 1969—Revised 2 May 1969)


Fig. 7. Cross-section between chromosomes and pole of an isolated anaphase spindle(spindle no. 4, Table 1). The enclosed area is shown at higher magnification in Fig. 8.x 6000.

Microtubule content of mitotic apparatus T75

tA * / t~S V_0 ' >»•. »• '

^ ^ • ^ ^

fa-^S.!.Fig. 8. Higher magnification view of the area delimited in Fig. 7. Anaphase spindle

cross-section, x 36000.


Fig. 9. Light-microscope view (Nomarski differential interference optics) of an average-sized, embedded metaphase mitotic apparatus prior to sectioning. This is mitoticapparatus no. I (Table 4).Fig. 10. Part of a cross-section through the aster of the mitotic apparatus shownin Fig. 9. Approximate location of the section within the mitotic apparatus is indicated.Microtubules appear singly (single arrow) as well as in groups of two or more (doublearrow). Radial density of microtubules in the aster is not as great as in the spindle,x 40000.

Fig. 11. Part of a cross-section through the spindle of the mitotic apparatus shownin Fig. 9. Approximate location of the section within the mitotic apparatus is indicated,x 40000.

an estimate of the amount of microtubule protein …procedure was the same as in (a), except that...

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