spindle birefringence of isolated mitotic apparatus ... · apparatus similar to one designed by...

J. Cell Set. 20, 3C9-327 (i976) 309Printed in Great Britain

SPINDLE BIREFRINGENCE OF ISOLATED

MITOTIC APPARATUS ANALYSED BY

PRESSURE TREATMENT

A. FORERBiology Department, York University, Dovmsview, Ontario MjJ 1P3, Canada

AND A. M. ZIMMERMANDepartment of Zoology, University of Toronto, Toronto, Ontario M$S 1A1, Canada

SUMMARY

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide mediumwere treated with pressure. Pressure treatment had no effect on spindle birefringence whenMA were in full-strength isolation medium. After placing MA in quarter-strength isolationmedium, pressures of 4-0 x io3— i-8 x 10* lbf in.~a (2-76 x IO4-I-24 x io6 kN m~s) for 15 mincaused reduction of birefringence which occurred in 2 steps: firstly 20-30% of the birefringencewas lost, and then, at higher pressures, the rest of the birefringence was lost. Electron micro-scopy suggested that pressure-induced changes were in non-microtubule material. Pressuretreatment had no effect on MA isolated with hexylene glycol when the MA were pressurizedin hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolatedin hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium andwere subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethyl-sulphoxide). We discuss the differences in response between isolated MA and in vivo MA,and we discuss the possibility that 2 components contribute to MA birefringence.

INTRODUCTION

Isolated mitotic apparatus (spindle-aster—chromosome complex) could be used tostudy the chemistry of chromosome movement if the isolated mitotic apparatus werefunctional (i.e. if anaphase chromosomes could be induced to move poleward). Butno one has yet reported chromosome movement in isolated mitotic apparatus (MA),although chromosome movement can continue when cells are gently lysed (Cande,Snyder, Smith, Summers & Mclntosh, 1974). One reason for the absence of chromo-some movement in the isolated MA might be that there are irreversible alterationsof MA components during the isolation procedure; for example, shortly after isolationof MA with hexylene glycol, MA microtubules decay, MA birefringence changes,and MA solubility properties change (e.g. Kane & Forer, 1965; Goldman & Rebhun,1969; Fulton, Kane & Stephens, 1971). We have described a new isolation procedureusing glycerol-dimethylsulphoxide (DMSO) as isolation medium, and have shownthat after isolation in this medium MA have stable microtubules, stable birefringence,and stable solubility properties (Forer & Zimmerman, 1974). The same mediumquantitatively preserves the number of polymerized cytoplasmic microtubules intissue culture cells (Rubin & Weiss, 1975). A disadvantage of the glycerol-DMSO

310 A. Forer and A. M. Zimmerman

isolation was the contamination of the MA preparation with cytoplasmic debris. Aprocedure for obtaining MA preparations with much less contamination is describedherein.

We have not yet been able to obtain chromosome movement in MA isolated inglycerol-DMSO despite the stable microtubules and apparent lack of denaturationduring isolation. Our main interest is in the mechanisms of chromosome movement;in trying to assess why we have not been successful in inducing chromosome move-ment in isolated MA preparations, we have compared isolated MA with in vivo MA,to see whether the two would respond in the same way to cold treatment and to pressuretreatment. (Similar comparisons have been made for the effects of cold treatment orof colchicine treatment on MA isolated by other methods: Inoue', Borisy & Kiehart,1974; Rebhun, Rosenbaum, Lefebvre & Smith, 1974.) Cold treatment of cells causescomplete dissolution of MA in vivo (e.g. Inoue", 1964), and pressure treatment of cells(6 x io3 lbf in.~2 (4-14 x io4 kN nv2) for 10 min) causes complete dissolution of MAin vivo (for review see Marsland, 1970; Zimmerman, 1970, 1971). We have studiedhow both these agents affect MA isolated in glycerol-DMSO, and in this reportdescribe the effects of pressure on MA birefringence and MA microtubules; in asubsequent article (Forer & Zimmerman, 1976) we describe the effects of cold treat-ment. Isolated MA do not respond to either treatment like MA in vivo.

MATERIALS AND METHODS

Sea urchins (Strongylocentrotus purpuratus or Lytecliimis pictus) were obtained from PacificBio-Marine Laboratories, Venice, California. Eggs and sperm were obtained as describedpreviously (Forer & Zimmerman, 1974). Mitotic apparatus (MA) were isolated in eitherM T M + EGTA (50% v/v glycerol; 10% v/v dimethylsulphoxide; 5 mM MgCl2; phosphatebuffer, at final pH = 6-8; and 0 1 mM EGTA), or in hexylene glycol (1 M hexylene glycol in001 M phosphate buffer, pH 60), as described previously (Forer & Zimmerman, 1974); theonly change was in the procedure for washing the MA, after the cells were lysed, as follows.

After lysis in M T M + EGTA (which we will designate MTME), the lysate was centrifugedat 225 g for 5 min. This pellet contained unlysed eggs, large pieces of cytoplasm, and someMA. The supernatant was then centrifuged at 3000 g for 15 min. This pellet, which containedmostly MA with little contaminating material, was resuspended in M T M E (rinse). (There wassome variation in the results; different batches gave somewhat different degrees of contamina-tion, so in some cases the low-speed centrifugation was repeated.) The MA were centrifugedand resuspended in new MTME (second rinse), and finally were centrifuged and resuspendedin MTME.

After lysis in hexylene glycol the lysate was placed in an ice bath and was centrifuged at2000 g for 15 min; the MA (pellet) were transferred to MTME (rinse). After one additionalrinse, the MA were resuspended and stored in MTME. Some aliquots were kept in hexyleneglycol, on ice.

For pressure treatment, MA were transferred to a small glass chamber (vol. ~ 1 ml), coveredwith Parafilm (kept tight with a rubber band), and placed in a stainless-steel pressure chamber(Landau & Thibodeau, 1962). The pressure chamber was connected to a temperature-pressureapparatus similar to one designed by Marsland (1950). Hydrostatic pressures up to i-8x io4

lbf in."', equivalent to 1-24 x io5 kN m~a, were applied to the cells with an Aminco pressurepump at the rate of 5 x io3 lbf in."1 (3'45 x 10* kN m~2) per stroke. The MA were treatedwith pressure at 20 °C, and for pressure treatment MA were suspended either in M T M E orin MTME diluted with P-Mg buffer (0-005 M MgCl2 in 0-006 M phosphate buffer, pH 6-8).When there was dilution of the MTME, aliquots of MA in M T M E were mixed with aliquotsof the diluent immediately prior to transfer to the glass chamber and to the pressure bomb.

Pressure-treated isolated MA 311

It required about 3 min from time of dilution of the MA to application of maximum pressure.The amount of dilution is described together with the results.

Birefringence was estimated by eye, using a A/30 rotating mica (Brace—K6hler) compensator,as described by Bennet (1950). Each MA was oriented at 45° to the crossed polars and thecompensator rotated until the MA appeared maximally dark; this was repeated after the MAwas rotated through 900, and the retardation was taken as one half of the difference in the 2readings (Goldman & Rebhun, 1969). The 'maximally dark' compensator position was takenas that compensator position where the most birefringent part of the MA became dark; for anMA isolated in MTM + EGTA, the most birefringent part of the MA is generally about midwaybetween the chromosomes and the poles, as described by Swann (1951) for in vivo MA inzygotes of a different species. There was some variation in stage of division in any preparationof MA, and we measured only MA which were at metaphase.

For electron microscopy, MA were fixed with glutaraldehyde (which was 2 % in the mediumin which the MA were found immediately prior to fixation). After varying times in glutar-aldehyde (ranging from hours to days), the MA were centrifuged into a pellet and then resus-pended in 1 % osmium tetroxide (which was 1 % in P-Mg). After 1 h the MA were centrifugedinto a pellet, and then mixed with molten 2% agar; after the agar had solidified, small pieceswere placed in 50% ethanol, dehydrated, rinsed with propylene oxide, and flat-embedded inEpon. Individual MA were located in the Epon, transferred to a block and sectioned, stainedand observed as described previously (Forer & Zimmerman, 1974). We transferred the MAinto molten agar after osmium tetroxide, because we found that the 'reversed contrast' imageof the MA microtubules described previously (Forer & Zimmerman, 1974) was due to the heatarising when the MA were transferred to the molten agar before the osmium tetroxide, whichwe did previously (Forer & Zimmerman, 1974).

Index-of-refraction measurements were made using an Abbe refractometer (Bausch andLomb, Rochester, N.Y.), used at the sodium yellow line.

Standard deviations were calculated as given previously (Forer & Goldman, 1972, table 1).To test for possible differences between distributions we used standard t-tables and calculatedthe parameter t as

t =/(s.p..:

V nx| (

where S and Fare the averages of the 2 sets of data, s.n.x and S.D., are the calculated standarddeviations of the 2 sets of data, nx and ny are the numbers of samples in each of the 2 sets ofdata, and nw + n, — 2 is the number of degrees of freedom in the t-table.

RESULTS

MA isolated in MTME

We previously established that isolated MA are stable for 2 weeks or more whenstored in MTME (Forer & Zimmerman, 1974). But there was no change in birefrin-gence when MA in MTME were treated with pressure, with pressure treatments ofi-8 x io4 lbf in.-2 (1-24 x io5 kN m~2) for 15 min. It was necessary to dilute theMTME in order for pressure to alter the birefringence, and for these experimentsthe dilution was the minimum necessary in order for cold treatment to alter MAbirefringence, namely quarter-strength MTME (Forer & Zimmerman, 1976); thedilution was achieved experimentally by mixing 1 part of MA suspended in MTMEplus 3 parts of P-Mg. Prior to studying the effects of pressure on MA birefringence,then, we also had to ascertain whether MA birefringence was stable in diluted MTME.

The MA birefringence changed when MA were placed in different media, andincreased by more than 50% when MA were diluted from MTME into quarter-


strength MTME (see Fig. i). Thus in studying possible effects of pressure we studiedMA after dilution into quarter-strength MTME, comparing non-treated MA(controls) with treated MA (experimentals).

The birefringence of control MA was not stable in quarter-strength MTME, butthere was not much change in birefringence during the first few hours after dilution(Forer & Zimmerman, 1976). Therefore, in order to minimize changes in MA bire-fringence not related to the experimental treatment, pressure treatment was begunwithin 3-5 min after dilution of the MA suspension, and all birefringence measure-ments (on both experimental and control MA) were completed within 45 min afterthe dilution. The results of the pressure treatments are as follows.

7

6

1 5

C

n"OL.

5 3

21

MTM/4

Ml

-

Mj2

v A1TA1

i i

liiyc

i i

1-34 1-36 1 38 1-40 1-42 1-44

Index of refraction

1-46 1-48

Fig. 1. Measured average retardations (closed circles) and standard deviations (verticallines) when 5. purpuratus MA originally isolated in MTME were transferred to newmedium. Each point is the average of readings of 20 MA. MA were initially in MTME(labelled MTM), and aliquots were diluted by 2, 4 or 8 with P-Mg (points labelledMTMjz, MTMI4. and MTM/8, respectively) and birefringences measured within20 min after dilution. Other aliquots were mixed 1 + 1 with glycerol (point labelledMTM/glyc), or pelleted MA were placed in pure glycerol (point labelled glyc). Theindices of refraction were measured on solutions free from MA. The dashed line is theleast-mean-squares fit to the points, while the solid line is a possible curve fitting thepoints. The 'in vivo' refractive index (arrow) was estimated assuming that 5.purpuratuszygote MA have the same concentration of dry matter in vivo as has been measuredinterferometrically in MA in vivo in other sea-urchin zygotes (Forer & Goldman,1972).

MA birefringence was reduced when the pressure exceeded a certain level; thereduction depended on the magnitude of the pressure and on the species of sea urchin(Figs. 2-4). The reduction of birefringence seemed to occur in 2 steps: in MA isolatedfrom S. purpuratus zygotes, for example, no birefringence was lost at pressures upto 6 x io3 lbf in.~2 (4-14 x io4 kN m~2); at pressures between 1 x io4 and 1-4 x io4 lbfin.~2 (6-89 x io4 and 9-65 x io4 kN m~2) about 20% of the MA birefringence waslost; finally, pressures above 1-4 x io4 lbf in.~2 (9-65 x io4 kN m~2) caused further loss

Pressure-treated isolated MA

of birefringence and eventually caused all of the MA birefringence to disappear (Fig.2). MA without any detectable birefringence were identified as MA by phase-contrastmicroscopy, for spindle structure and chromosomes remained clearly visible. Pressuretreatment also caused reduction in birefringence of MA isolated from L. pictus zygotes(Fig. 3), but the low pressure and high pressure ' cut off' points were at lower pressuresthan for MA isolated fiom S. purpuratus zygotes (Fig. 4).

120

100

80

60

40

20

i

I I I

0 2 4 6 8 10 12 14 16 18Pressure, Ibf In.- 'x 10"1

1 1 I I

345 6-90 10 35Pressure, kN r r r ' x iO- 4

Fig. 2. The effects of 15-min pressure treatment on 5 . purpuratus zygote MA isolatedin MTME, and diluted into quarter-strength M T M E immediately before pressuretreatment. The circles represent average values, and the vertical lines extending fromthe circles are the calculated standard deviations. Each circle is the average of 20readings. The closely spaced points at 6 x 10s, 8 x io! , and 16 x io ' lbf in.~* (4-14 x 10*,5-56 x io4, and 11 02 x io4 kN m"1) are repeats of the same treatments done on 2different days. The dashed line represents the approximate range of statisticallydifferent values (at P < o-oi) as calculated using the t test. The solid line is drawnhorizontally when the points in question are statistically not different in birefringence.The probability that the MA birefringences measured after 8 x 10' lbf in."1 (5-56 xio4 kN m~*) are the same as the birefringences of the control MA is 001 < P < 005 ;at higher pressures the probabilities that the MA birefringences measured afterpressure treatment are the same as the birefringences of the control MA are allP << o-ooi.

The storage of MA (at room temperature) for 2 weeks after isolation did not alterthe results described above; when the MA were transferred to quarter-strengthMTME at various times after isolation and treated with a specific pressure, the samepercentage loss of birefringence was obtained as with freshly isolated MA.

Pressure-treated S. purpuratus zygote MA were studied electron microscopically,to determine which components were altered by the pressure treatment. We studiedisolated MA (a) from the controls, (b) from treatment at 1-4 x io4 lbf in."2 (9-65 x io4

kN m~2) which caused a measured loss of 30 % of the birefringence, and (c) fromtreatment at i-6x io4 lbf in."2 (I-IO x io5 kN m~2) which caused a measured loss of

A. Forer and A. M. Zimmerman

o 1 2 0

100

80

60

40

20

\

ATM 4 6 8 10

Pressure, Ibf in.-Jx10"a

12 14

3-45 6-90

Pressure, kN rrTJx10-4

10-35

Fig. 3. The effects of 15-min pressure treatments on L. picttis zygote MA isolated inMTME and diluted into quarter-strength MTME immediately before pressuretreatment. The circles are average values, the lines extending from the circles arestandard deviations, and each circle is the average of 20 or more readings. The prob-abilities that the MA birefringences measured after pressure treatments of 6 x io3 Ibfin."1 (414 x io4 kN m~a) or greater are the same as the birefringences of the controlMA are all P < o-or.

120

100

80

60

40

20

-

V

--

1 I 1 1 1 1

\\

\N

\

\\

\\

\

6 8 10 12Pressure, Ibf in.- 'x 10"1

1 1

14 16 18

1

3-45 6-90Pressure, kN m-Jx10-4

10-35

Fig. 4 consists of the curves of Fig. 2 (S. purpuratus, dashed line) and Fig. 3 (L. pictus,solid line) plotted on the same graph, to illustrate the similarities and differences(described in the text).


70 % of the birefringence. Serial sections of at least 2 MA from each category wereanalysed, and we were unable to discern any differences in the approximate numbersand distribution of microtubules in the 3 groups of MA (Figs. 5-13). Nor couldcolleagues who were given non-labelled sets of micrographs of the MA decide fromthe numbers of microtubules which MA were treated and which were controls. How-ever, there was a difference in non-microtubule MA material. In control MA therewas granular material associated with the microtubules (as described by Goldman &Rebhun, 1969); the electron-dense 'granules' appeared roundish and discrete, about20 nm in diameter, and the microtubule wall was clearly discernible (Figs. 8, 10).MA treated with i-4x io4 lbf in.-2 (9-65 x io4 kN m-2) appeared like control MA(compare Figs. 5, 8 and 10 with Figs. 6, 12 and 13). In the MA treated with i-6x io4

lbf in.-2 (I-IOX io5 kN m"2), on the other hand, discrete granules and readily dis-

cernible microtubule walls were only rarely seen. Instead there was amorphous,darkly staining material associated with the microtubules (Figs. 9, 11), the walls ofwhich were often difficult to see clearly. In MA treated (inadvertently) with heat afterglutaraldehyde fixation (see Materials and methods section) there was also darklystaining, amorphous material associated with the MA microtubules (Forer & Zimmer-man, 1974); this similarity in appearances suggests that both pressure and heat treat-ments cause alterations in a non-microtubular MA component.

MA isolated in hexylene glycol

S. purpuratus zygote MA were isolated in hexylene glycol and stored on ice for 3 hprior to pressure treatment at i-8 x io4 lbf in.~2 (1-24 x ioB kN nv~2) for 15 min. Wecould detect no difference between the birefringence of the control MA and thepressure-treated MA. This is in contrast to the total loss of birefringence seen afterMA isolated in MTME were treated with the same pressure (Fig. 2).

Other S. purpuratus zygote MA were isolated in hexylene glycol, transferred to

Table 1. Effects of pressure on S. purpuratus zygote MA

lbf in.-'x

ci-o1-2

1 6i-8

Pressureio4 kN m~* x io4

0

6898 2 8

11 02

12-41

Birefringenceas

remaining after. % of control

t

MA isolated in hexylene glycoland transferred

shortly after :

ioo± 158 8 ± i 3 ( «8 3 ± i 6 ( n40 ± 17 (n

0

to MTMEisolation

= 20)

= 2C)

= 20)

pressure,

MA isolatedin MTME

(data of Fig. 2)

1 0 0

80

7738-46

0

All pressure treatments were for 15 min, starting within a few minutes after MA were dilutedinto quarter-strength MTME. Some of the tabulated data are given as average values ± standarddeviations, where n gives the numbers of MA measured. All pressure treatments tabulatedresulted in birefringences different from the controls at levels of significance of P =S o-oi(determined using the t test).


MTME shortly after isolation, and stored at room temperature. After storage forfrom 3 h to 6 days the MA were diluted into quarter-strength MTME and treatedwith pressure. Pressure treatment caused loss of birefringence, which seemed to occurin 2 steps (Table i).

It might be relevant to note that MA isolated in hexylene glycol and transferredimmediately to MTME had (on average) 20 % less birefringence than MA isolated inMTME. Similarly, they had 20% less birefringence when diluted into quarter-strength MTME than MA isolated in MTME and diluted into quarter-strengthMTME. Thus, while the percentage changes in birefringence might be similar in the2 columns of Table 1, the absolute values of the control birefringences differ.

DISCUSSION

MA isolated in MTME seem to respond to pressure treatment differently than doMA in vivo. Whereas MA in vivo disappear after pressure treatment of around 4 x io3-8 x io3 lbf in.~2 (2-76 x io4~5-52 x io4 kN m~2) (Pease, 1946; Marsland, 1956; Zim-merman & Marsland, 1964; Salmon & Ellis, 1975; Salmon, 1975 a), in what appears tobe a one-step loss of spindle structure and of spindle birefringence, isolated MA losebirefringence in 2 steps. The first step is a loss of around 25 % of the birefringenceand the last step (at distinctly higher pressures) is loss of the remaining birefringence.The difference between isolated MA and in vivo MA might indicate that there is afunctional alteration during isolation. If one wants to achieve chromosome movementin isolated MA then one should try to obtain isolated MA which respond to pressureas do in vivo MA.

One possible reason for the difference between MA isolated in MTME and in vivoMA might be that some MA component is 'denatured' during the isolation. Theinitial 25 % loss of birefringence in isolated MA occurs at pressures near those whichcause complete loss of birefringence of MA in vivo (Salmon, 1975 a); thus one assumesthat the higher pressures necessary to cause complete loss of birefringence are neededto solubilize the denatured component. Alternatively, all MA components might benative, but the 12-5 % (or 1-7 M) glycerol present in quarter-strength MTME mightprotect an MA component against pressure. If the former explanation is correct, thenin trying to get isolated MA to respond like in vivo MA one needs to change theisolation method; if the latter is correct then one needs to change the condition underwhich the pressure is applied. It is relevant to note that Sakai & Kuriyama (1974)have shown that 4 M glycerol stabilizes otherwise unstable MA isolated in a 1 Mglycerol isolation medium; also, Salmon (1975 b) has shown that 15% glycerol protectsrepolymerized brain microtubules against the effects of pressure.

Regardless of which explanation is correct, that there are 2 discrete steps in theloss of birefringence suggests that 2 components contribute to the birefringence.These may be 2 quite different components (such as microtubules and another com-ponent), or 2 different classes of the same component (such as 2 classes of microtubules,which respond differently to the isolation procedure or to pressure treatment inquarter-strength MTME). Our data suggest the former possibility, namely that a

Pressure- treated isolated MA 317

non-microtubule component contributes to the birefringence, for, electron micro-scopically, there were no readily apparent differences between control MA and MAtreated with i-4x io4 lbf in.~2 (9-65 x io4 kN m~2) which lost 30% of their bi-refringence, whereas pressure-treated MA which lost 70 % of their birefringence hadapproximately normal numbers of microtubules, and had alteration in a non-microtubule component (giving rise to amorphous, darkly staining material). Becauseof the nature of the birefringence measurements, however, without serial-sectionreconstruction of MA we cannot rule out the possibility that 2 classes of microtubulesare responsible for the 2-step pressure sensitivity, as follows.

If there were an overall drop of 70 % in the numbers of microtubules, we coulddetect this, even without serial-section reconstruction. However, a drop in 70% ofthe measured birefringence does not necessarily correspond to a drop in 70 % of thebirefringence throughout the entire MA: we measured birefringence of the single MAregion which was maximally birefringent, and a change in the region which wasmeasured may or may not be accompanied by corresponding changes in other regionsof the same MA. If, for example, the measured region were twice as birefringent asother regions, then a 70 % drop in the birefringence of the measured region might beaccompanied by zero change in the birefringence of other regions. One could imagine,for example, that solely microtubules gave rise to birefringence, and that pressuretreatment caused loss of 'free microtubules' (i.e. those in which both ends terminatein the half-spindle, and which are connected neither to kinetochore nor to pole, asdefined by Mclntosh et al. 1975, and as also identified by Heath, 1974); this could givea local change of 70 % of the birefringence with little noticeable change elsewhere inthe MA. In this case one would expect no overall change in numbers of microtubules,but rather only local changes; one would need serial-section reconstruction of theMA microtubule dispositions to detect such local changes. Data argue against thispossibility, for qualitatively, anyway, the pressure treatment seems to cause loss ofbirefringence throughout the entire MA, and furthermore, pressure treatment doescause alterations in non-microtubular MA components; but we cannot definitivelyrule out the possibility that 2 classes of microtubules give rise to the 2-step responseto pressure.

It is relevant to point out that whereas pressures of 4 x io3-8 x io3 lbf in.~2 (276 xio4-5'52x io4kN m~2) often cause loss of spindle or cytoplasmic microtubules (e.g.Tilney, Hiramoto & Marsland, 1966; Kennedy & Zimmerman, 1970), pressure treat-ments of 1 x io4 lbf in.""2 (6-89 x io4 kN m~2) do not cause loss of neurotubules in vivoor in vitro (O'Connor, Houston & Samson, 1974), and do not cause loss of fungalspindle microtubules in vivo (Heath, 1975); nor does a pressure treatment which causesspindle breakdown in meiosis I necessarily cause spindle breakdown in meiosis II orin mitosis in the same species (Pease, 1946). Thus, different microtubules mightquite well differ in their susceptibilities to pressure.

High-pressure treatments which did not cause loss of neurotubules in vivo did affectnon-microtubular material in axons and olfactory nerves, for after pressure treatmentthere was an increase in electron density of the 'cytoplasmic matrix' (O'Connor et al.1974). Perhaps this is similar to the amorphous, electron-dense material we saw


around the microtubules after isolated MA were treated with i-6x io*lbf in.~2

(I-IO x JO5 kN m~2) for 15 min (Figs. 8-11).We considered the possibility that the 20% difference in birefringence between

MA isolated in MTME and MA isolated in hexylene glycol was due to materialpreserved during MTME isolation which was not preserved during hexylene glycolisolation, and that perhaps this 'extra' material caused the initial 20% drop in bire-fringence after low-pressure treatments. If this were true, then the pressure effect onMA isolated in hexylene glycol should be a single-step effect, i.e. without the first20% loss of birefringence. This possibility does not seem to be correct, however,since birefringence in the hexylene glycol-isolated MA seems to be reduced bypressure in 2 steps (Table 1).

A large part of the birefringence of MA isolated in MTME seems to be form bire-fringence, as evidenced by Fig. 1: increasing the index of refraction of the imbibingmedium reduced the MA birefringence by at least 70 % from the estimated in vivobirefringence. Similar conclusions were reached previously on hexylene glycol-isolated MA which were fixed with osmium tetroxide and then dehydrated (Rebhun& Sander, 1967). Our data extend the earlier experiments with respect to severalpoints. Firstly, our data are on MA with higher initial birefringence (MA isolated inhexylene glycol have only 80 % of the birefringence of those isolated in MTME), andthus would seem to better represent MA in vivo. Secondly, our data are on unfixedMA, whereas the fixation procedure caused some loss of birefringence (Rebhun &Sander, 1967). Finally, our data are on the MA proteins themselves, whereas theprevious data were on protein—osmium complexes (for the MA turned brown orblack after fixation (Rebhun & Sander, 1967)).

While one might conclude from our data that 70 % or more of the birefringence ofisolated MA is form birefringence, we should point out that this conclusion reallyapplies only to the region which we measured (the region of maximum birefringence),and, as discussed previously, the same conclusion does not necessarily apply to otherregions of the same spindles. Nor does the same conclusion necessarily apply to MAin vivo.

We should also point out that the differential centrifugation method for cleaningup the preparation of MA isolated in MTME has yielded MA pellets clean enough forchemical analysis, which is a distinct improvement over our previously publishedmethod.

We acknowledge with appreciation the skilful sectioning of Mary Lou Ashton. This workwas supported by grants from the National Research Council of Canada.

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CANDE, W. Z., SNYDER, J., SMITH, D., SUMMERS,- K. & MCINTOSH, J. R. (1974). A functionalmitotic spindle prepared from mammalian cells in culture. Proc. natn. Acad. Sci. U.S.A. 71,1559-1563-


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FORER, A. & ZIMMERMAN, A. M. (1974). Characteristics of sea-urchin mitotic apparatus isolatedusing a dimethylsulphoxide/glycerol medium. J. Cell Sci. 16, 481-497.

FORER, A. & ZIMMERMAN, A. M. (1976). Spindle birefringence of isolated mitotic apparatusanalysed by treatments with cold, pressure, and diluted isolation medium. J. Cell Sci. 20,329-339-

FULTON, C , KANE, R. E. & STEPHENS, R. E. (1971). Serological similarity of flagellar andmitotic microtubules. J. Cell Biol. 50, 762-773.

GOLDMAN, R. D. & REBHUN, L. E. (1969). The structure and some properties of the isolatedmitotic apparatus. .7. Cell Sci. 4, 179—209.

HEATH, I. B. (1974). Mitosis in the fungus Thraustotheca clavata. J. Cell Biol. 60, 204-220.HEATH, I. B. (1975). The effect of antimicrotubule agents on the growth and ultrastructure of

the fungus Saprolegnia ferax and their ineffectiveness in disrupting hyphal microtubules.Protoplasma (in Press).

INOUE, S. (1964). Organization and function of the mitotic spindle. In Primitive Motile Systemsin Cell Biology (ed. R. D. Allen & N. Kamiya), pp. 549—598. New York: Academic Press.

INOUE, S., BORISY, G. G. & KIEHART, D. P. (1974). Growth and lability of Chaetopterus oocytemitotic spindles isolated in the presence of porcine brain tubulin..?. Cell Biol. 62, 175-184.

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(Received 7 August 1975)

Figs. 5-7. Low-power electron micrographs of a control MA (Fig. 5), an MA treatedwith 14 x io4 lbf in.~* (965 x io4 kN m~f) for 15 min, after which the birefringencewas reduced by 30 % (Fig. 6), and an MA treated with i-6 x 10* lbf in."1 (I-IO x ic6 kNm~a) for 15 min, after which the birefringence was reduced by 70% (Fig. 7). ControlMA were not treated with pressure, but were diluted into quarter-strength MTMEat the same time as the pressure-treated MA and were fixed with glutaraldehyde at thesame time as were the pressure-treated MA. The same MA are illustrated in higher-power micrographs in Figs. 8—13. x 5200.


Figs. 8, 9. Intermediate-magnification electron micrographs of a control MA (Fig. 8),and of an MA treated with i-6 x io4 Ibf in."2 ( I - IO x io6 kN m~s) for 15 min (Fig. 9),illustrating the large numbers of microtubules in both MA. Some microtubules areindicated by arrows, x 17600.


Figs. 10, 11. Higher-power electron micrographs of a control MA (Fig. 10) and ofan MA treated with 16 x io4 lbf in.-1 (110 x io5 kN m"2) for 15 min (Fig. n ) , illus-trating the appearances of both the microtubules and the non-microtubular materialin the MA. Some microtubules are indicated by arrows. The non-microtubular materialappears altered by the pressure treatment; it appears to be more diffuse and toaggregate near the microtubules. The insets illustrate cross-sectioned microtubules(from the astral regions) showing that in the control MA the cross-sectioned micro-tubules appear with normal-appearing black walls, whereas in the pressure-treatedMA the cross-sectioned microtubules appear with walls with reversed contrast,similar to those we described previously (Forer & Zimmerman, 1974). X500CO.Inset to Fig. 10, x 78500; to Fig. 11, x 163000.


Figs. 12, 13. Electron micrographs of an MA treated with 14 x io4 lbf in."1 (9-65 x io4

kN m"1) for 15 min, illustrating the large numbers of microtubules (Fig. 12), and theappearances of the microtubules and of the non-microtubular material (Fig. 13).Some microtubules are indicated by arrows. Both the microtubules and the non-microtubular material appear the same as in the control cells (compare Figs. 8 andic). Fig. 12, x 30200; Fig. 13, x 67500.

Pressure-treated isolated MA

spindle birefringence of isolated mitotic apparatus ... · apparatus similar to one designed by...

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