Characteristics of sea-urchin mitotic apparatus isolated using a dimethyl sulphoxide/glycerol medium

Our understanding of mitosis could be greatly advanced by the study of chromo some movement in vitro. The first step toward this goal was the mass isolation of the mitotic apparatus (i.e. the spindle-aster-chromosome complex); sea-urchin zygotes were stabilized with cold ethanol and then treated with digitonin to release the mitotic apparatus (Mazia & Dan, 1952). While the mitotic apparatus (MA) in vivo are labile, the MA isolated with ethanol-digitonin were not very soluble. Thus, isolation methods were subsequently developed which resulted in MA which were more easily soluble and which, because they were easily soluble, presumably were more like in vivo MA. The newer methods for isolating sea-urchin zygote MA developed by Mazia and co-workers utilize as lysing and stabilizing agents dithiodiglycol (Mazia, Mitchison, Medina & Harris, 1961), or dithiodipropanol (Sakai, 1966), or, most recently, replacement of the Na+ in seawater with Li+, followed by storage in cold ethanol and lysis in a Triton X-ethanol solution (Mazia, Petzelt, Williams & Meza, 1972). Other isolation methods utilize as lysing and stabilizing agents either hexanediol (Kane, 1962a) or hexylene glycol (Kane, 1965), or tubulin-polymerizing medium (Rebhun, Rosenbaum, Lefebvre & Smith, 1974). Whilst the MA isolated with all of these techniques have been used to study MA chemistry (e.g. Mazia & Dan, 1952; Went, 1959; Zimmerman, 1960; Miki-Noumura, 1965, 1968; Sakai, 1966, 1968; Wilt, Sakai & Mazia, 1967; Kane, 1967; Stephens, 1967; Hartmann & Zimmerman, 1968, 1971; Weisenberg & Taylor, 1968; Mazia et al. 1972), factors governing stability and size of MA (e.g., Mazia & Dan, 1952; Mazia & Zimmerman, 1958; Mazia, Harris & Bibring, 1960; Kane, 1962a, 1965; Marsland & Zimmerman, 1965; Cohen, 1968; Goode & Roth, 1969; Goode, 1973), MA microtubules (e.g. Kane, 1962b; Kane & Forer, 1965; Kiefer, Sakai, Solari & Mazia, 1966; Bibring & Baxandall, 1968, 1971; Cohen & Rebhun, 1970; Cohen & Gottlieb, 1971; Fulton, Kane & Stephens, 1971), MA birefringence (e.g. Kane & Forer, 1965; Rebhun & Sander, 1967; Goldman & Rebhun, 1969; Bryan & Sato, 1970; Stephens, 1972), MA dry mass (Rustad, 1959; Forer & Goldman, 1969,1972), and the cold lability and incorporation of brain tubulin into isolated MA (Rebhun et al. 1974), chromosome movement has not occurred in isolated MA. Thus one of the main goals, obtaining chromosome movement in vitro, has not yet been achieved in isolated MA (see reviews: Hartmann & Zimmerman, 1974; Forer, 1969; Nicklas, 1971).

MA as presently isolated are not suitable in several other respects besides the inability to allow chromosome movement. Isolated sea-urchin zygote MA contain only 10% of the material present in in vivo MA (Forer, 1969; Forer & Goldman, 1972). Further, of the measurable properties of MA isolated from marine eggs, the birefringence rapidly decays to a final value of between zero and 50% of the initial birefringence (Kane & Forer, 1965; Rebhun & Sander, 1967; Goldman & Rebhun, 1969; Forer & Goldman, 1969; Bryan & Sato, 1970); the decay takes several hours at room temperature or 1 – 2 days at o °C, though storage in 2 M sucrose seems to prevent completely the birefringence decay (Goldman & Rebhun, 1969). The microtubules disappear, too, with about the same speed as the birefringence decays (Kane & Forer, 1965; Goldman & Rebhun, 1969). In addition the solubility properties change: whereas freshly isolated hexylene glycol MA are soluble in 0 · 5 M KC1, stored MA are not (Kane & Forer, 1965; Fulton et al. 1971), and these changes in solubility occur very quickly after isolation, for striking differences in solubility can be seen between freshly isolated Echinus zygote MA (Forer & Goldman, 1969) and those stored at o °C for 4 – 5 h (A. Forer, in preparation). Similarly, MA isolated in tubulin-polymerizing medium lose their cold lability within minutes (Rebhun et. al. 1974).

One approach to stabilizing MA microtubules is suggested by recent investigations of Filner & Behnke (1973, 1974), who described a method in which brain microtubules, which are labile in vivo, were stabilized and isolated as such in a medium containing 50% glycerol, 10% DMSO (dimethyl sulphoxide), 5 HIM MgCl₂, and 5 mM phosphate buffer (Filner & Behnke, 1973, 1974), this medium being called MTM (microtubule medium). We adapted their method to isolating MA from sea-urchin zygotes: as reported herein, the resultant isolated MA have stable birefringence, stable micro-tubules, and stable solubility properties, as well as an increased concentration of dry matter.

Sea urchins (Strongylocentrotus purpuratus) obtained from Pacific Bio-Marine Laboratories, Venice, California, were kept at 12 °C and maintained for up to 3 months in artificial seawater (Instant Ocean, Aquarium Systems Inc., Wickliffe, Ohio). Eggs were obtained by injection of 0 · 53 M KC1 (Harvey, 1956), and were rinsed in several changes of seawater prior to use. Sperm was obtained from excised testes stored at 4 °C; diluted sperm solutions were freshly prepared prior to use.

For isolations of MA, fertilization membranes were removed using1 M urea, pH 7 · 8 (Kane, 1962 a). Zygotes with fertilization membranes removed were placed in 18 °C seawater until metaphase of the first post-fertilization division, which occurred approximately 75 – 85 min after insemination. The metaphase cells were rinsed with the isolation medium and then centrifuged gently (using a hand-powered centrifuge). After discarding the supernatant, the cells were resuspended in about 10 times their volume of isolation medium, in a 50-ml round bottom test tube, and this mixture was agitated strongly using a vortex mixer; this agitation resulted in cell lysis and liberation of intact MA. The isolation procedures were conducted at room temperature except for MA isolated using hexylene glycol: the hexylene glycol MA were placed in an ice bath immediately after the zygotes were lysed and all remaining steps were done at 0°C. After cell lysis the MA were centrifuged at 500 g for 5 min. The MA pellet was resuspended in isolation medium and this (rinse) step repeated once more.

We confirm the report by Filner & Behnke (1974) that bacteria do not grow in MTM. Thus, MA could be stored for months, at room temperature, without danger of bacterial contamination, even without addition of antibiotics.

The compositions of the isolation media were: (1) MTM: 50% (v/v) glycerol, 10% (v/v) dimethyl sulphoxide (DMSO), 5 mM MgCl2, 5 mM Sorensen’s phosphate buffer, final pH 6-8. (2) MTM without Mg2+: 50% (v/v) glycerol, 10% (v/v) DMSO, 5 mM Sorensen’s phosphate buffer, final pH 6-8. (3) MTM + EGTA: 50% (v/v) glycerol, 10% (v/v) DMSO, 5 mM MgCl₂, 5 mM S ø rensen’s phosphate buffer, final pH 6 · 8, and 0· 1mMEGTA [ethyleneglycol-bis (β-aminoethyl ether) N,N’-tetra-acetic acid]. (4) MTM + EGTA, pH 6 · 0: same as (3), but pH adjusted to 6 · 0. (5) Hexylene glycol, pH 6 · 4: 1 M hexylene glycol (2-methyl-2,4-pentane diol), 0 · 01 M S ø rensen’s phosphate buffer, final pH 6 · 4. (6) Hexylene glycol, pH6 · o: same as (5), but pH adjusted to 6 · 0. All pH adjustments were with HC1 or NaOH, as necessary.

Standard phase-contrast and polarization microscopical methods were used. Birefringence (retardation) was estimated by eye, judging the maximum darkness of the MA using either the Senarmont method, or the rotating mica (Brace-Kohler) method, both of which are described by Bennett (1950).

Index of refraction measurements were made using an Abb6 refractometer (Bausch and Lomb, Rochester, N.Y.).

For electron microscopy, MA were fixed for several hours in room temperature glutaraldehyde (2% in whichever medium the MA were in); then the MA were pelleted and placed in molten 2% agar. After the agar solidified, pieces of agar (containing MA) were fixed overnight in glutaraldehyde, postfixed for 1 h in osmium tetroxide (1% in the same medium used for glutaraldehyde), rinsed with H2O, kept overnight in 1% uranyl acetate in H2O, and dehydrated with increasing concentrations of ethanol. After 2 changes of propylene oxide the specimens were embedded in Epon, using standard methods (Glauert, 1965). Sections were cut on a Porter-Blum MT-2 microtome with a diamond knife and stained first with aqueous uranyl acetate (Watson, 1958) and then with lead citrate (Fiske, 1966). The sections were examined in a Philips EM 201 microscope, operated at 60 kV.

The MA isolated in various isolation media were compared using phase-contrast microscopy.

MA were present when zygotes were lysed either in MTM or in MTM without Mg²⁺, but those MA isolated without Mg²⁺ were markedly inferior, using the following criteria. The MA isolated in MTM (i.e. with Mg²⁺) had distinctly fibrous spindles and asters, chromosomes were visible, and spindles and asters were about the same density, as observed using phase-contrast microscopy. Those isolated in MTM without Mg²⁺, on the other hand, did not have visible fibres in the spindle or aster region, chromosomes were often not visible, and the MA were of low contrast, with both the centres of the astral regions and the spindles being of much lower density than other areas. Therefore, MTM (with Mg²⁺) was used for subsequent isolations.

Suitable MA were isolated using MTM, but the zygotes were often very difficult to lyse, and the preparations invariably contained clumps of cytoplasm which were about the same size as the MA. Working on the supposition that intracellular Ca²⁺ may have been responsible for the cytoplasmic clumping, EGTA, an agent known to sequester Ca²⁺ preferentially, was added to the MTM. When MTM + EGTA was used zygotes were easier to break and there were fewer large pieces of cytoplasm present. Therefore MTM + EGTA was used for subsequent isolations.

MA isolated in MTM + EGTA at pH 6-8 were compared with MA isolated in MTM + EGTA at pH 6-o, because it is known for other isolation media that the stability and appearance of MA depend on the pH (Kane, 1962a, 1965; Forer & Goldman, 1969). The MA isolated at pH 6 · 0 were refractile, with a large amount of surrounding cytoplasm, and, in contradistinction to isolations at pH 6 · 8, at pH 6 · 0 the cytoplasm was very poorly dispersed. Therefore MTM + EGTA (at pH 6 · 8) was used for subsequent isolations.

Zygotes consistently and reliably lysed in hexylene glycol; parallel isolations with MTM + EGTA seemed to yield fewer MA and less consistent lysis than isolations with hexylene glycol, for there were generally more unbroken zygotes contaminating the MTM + EGTA preparations than the hexylene glycol preparations. Furthermore, the lysis with hexylene glycol caused the cytoplasm to be dispersed into very small pieces, whereas the lysis with MTM + EGTA generally did not disperse the cytoplasm as well, and pieces of cytoplasm the same size as the MA were not uncommon. These were the two biggest disadvantages of the MTM + EGTA isolation method compared with the hexylene glycol method.

The MA isolated by the 2 methods differed markedly in appearance. Clear, linear spindle fibres were visible in the spindle and astral areas of MA isolated in MTM + EGTA (see Fig. 1); few clear linear fibres were seen in MA isolated in hexylene glycol at either pH (see Fig. 2). On the other hand, chromosomes were of much less contrast in MA isolated in MTM + EGTA than were chromosomes in MA isolated in hexylene glycol (compare Figs. 1 and 2). This is not due to the MTM + EGTA being of different refractive index than the hexylene glycol, for when MA isolated in hexylene glycol were subsequently transferred to MTM + EGTA the chromosomes were still seen much more clearly (and were of much more contrast relative to the spindle) than were chromosomes in MA isolated originally in MTM + EGTA. From the difference in contrast between the chromosomes and the spindles in the 2 kinds of MA we deduce (in the Discussion) that MA isolated in MTM + EGTA have a higher concentration of dry matter than those isolated in hexylene glycol.

It is relevant to point out that MA in vivo appear similar to MA isolated in MTM + EGTA but not to MA isolated in hexylene glycol. That is to say, chromosomes are not seen clearly, if at all, in sea-urchin zygote MA in vivo studied by phase-contrast or quantitative interference microscopy (Forer & Goldman, 1972, and in preparation). This is similar to the weak contrast of chromosomes in MA isolated in MTM + EGTA but distinctly different from the clearly visible chromosomes in MA isolated in hexylene glycol.

MA were generally stored in MTM + EGTA at room temperature, but storage at 0°C had no apparent effect on the MA as viewed with phase-contrast microscopy.

We judged the retardations of MA by eye. The retardations obtained by this method both can be erroneously high (Ross, 10,67;Pluta, 1969, 1971, 1972; Forer & Goldman, 1972) and can vary from observer to observer (Ross, 1967; Pluta, 1969; A. Forer & R. D. Goldman, unpublished). Thus the retardations measured in the present study should be considered as only ‘semiquantitative’. Nonetheless, the method is reliable for determining if changes in birefringence occur and is reliable for comparing retardations of MA isolated in different ways.

MA isolated in MTM + EGTA had initial retardations of 2· 5 – 3· 5 > as measured in 8 separate isolations. (There was more variation between MA of different isolations than between MA in any given isolation.) The MA retardations did not change during storage for at least 3 weeks when MA were stored at room temperature in MTM + EGTA. Nor were there any measurable changes when the MA were stored at o °C. Thus, in contradistinction to MA isolated in hexylene glycol (Kane & Forer, 1965; Bryan & Sato, 1970), MA isolated in MTM + EGTA have stable birefringence.

It is conceivable, however, that the stable retardations of the MA isolated in MTM + EGTA might represent only a fraction of the birefringence of MA in vivo; this stable birefringence, then, might be similar to the stable level of ‘residual’ birefringence found in isolated MA stored for extended periods in hexylene glycol which represents only 25 – 50% of the original birefringence (Rebhun & Sander, 1967; Goldman & Rebhun, 1969; Forer & Goldman, 1969; Bryan & Sato, 1970). To test this possibility one could compare the retardations of isolated MA with those in vivo to see if the stable retardations of MA isolated in MTM + EGTA are lower than those of MA in vivo. A less direct method is required in the present case, however, because, unfortunately, living S. purpuratus zygotes contain refractile granules which obscure the MA. And even if one could measure birefringence of in vivo MA, the retardations of isolated MA might not be the same as the retardations of in vivo MA, despite complete preservation of the birefringent material, depending on (1) how many components contribute to the birefringence of MA in vivo and whether the contributions are of form or intrinsic birefringence; (2) the amount of mass preserved during the isolation procedures, both of the oriented, birefringent elements and of their surrounds; and (3) the index of refraction of the medium in which the MA are suspended. We have data only on the last of these for there are no unequivocal data on the first points, and we have no quantitative data on the second point for the MTM + EGTA isolations. For these reasons as well it is necessary to use an indirect approach to the comparison of isolated MA with in vivo MA.

If it is assumed that form birefringence is a major part of the observed spindle birefringence (e.g. Rebhun & Sander, 1967), then the spindle retardation is expected to be different when spindles are in media of different refractive indices. Goldman & Rebhun (1969) have shown this to be true for MA isolated in hexylene glycol; the MA birefringence was reduced when MA were transferred to 2 M sucrose, and was increased when the MA were resuspended in hexylene glycol. Our results are similar. MTM + EGTA has a measured refractive index n_D= 1 · 423, and the hexylene glycol isolation medium has a measured refractive index n_D = 1 · 348. When MA isolated in MTM + EGTA were transferred to hexylene glycol the MA increased in retardation by about 60% (for example, from 3 to 4-8 nm). Using this information on measured MA retardations after transfer to hexylene glycol, one can indirectly determine how the retardations of MA isolated in MTM + EGTA compare with those of MA in vivo, as follows.

Retardations of Echinus and Psammechinus zygote MA isolated in hexylene glycol under various conditions have been compared with retardations of Echinus and Psam mechinus zygote MA in vivo (Forer & Goldman, 1969, and in preparation); when isolated in solutions of appropriate pH, the MA have the same retardations as MA in vivo. Assuming that these results apply to zygotes of other species of sea-urchin as well, one can indirectly estimate the retardations of Strongylocentrotus zygote MA in vivo by measuring the retardations of Strongylocentrotus zygote MA isolated in hexylene glycol. In doing this one must first choose hexylene glycol at an appropriate pH, for the retardations of MA isolated in hexylene glycol are the same as in vivo only when the pH of the isolation is more acid than usual (Goldman & Rebhun, 1969; Forer & Goldman, 1969; A. Forer & R. D. Goldman, in preparation). To complicate matters, one is not justified in comparing the solutions at the same pH, because with different species one may get MA with equivalent physiological properties only at different pH values (Forer & Goldman, 1969). Thus, one must determine anew the pH properties of MA from each new species. In the present study, Strongylocentrotus zygote MA isolated using hexylene glycol at pH 6 · 0 and 6 · 4 were roughly equivalent in properties to Echinus zygote MA isolated at pH 6 · 1 and 6 · 8 (described by Forer & Goldman, 1969), using the following criteria: (1) the MA isolated at lower pH had measurable residual birefringence, while the MA isolated at high pH had none; and (2) the low pH MA were soluble only in the spindle region, while the high pH MA were completely soluble. Since MA isolated from Echinus zygotes using hexylene glycol at pH 6 · 1 had about the same retardations as MA in vivo (Forcr & Goldman, 1969), the equivalent Strongylocentrotus MA (isolated in hexylene glycol at pH 6 · 0) can be used as indicators of the approximate retardations of Strongylocentrotus zygote MA in vivo. MA isolated in hexylene glycol at pH 6 · 0 had maximum retardations of 4 · 5 nm. MA isolated in MTM + EGTA (pH 6 · 8) had maximum retardations of 3 · 5 nm, and following transfer to hexylene glycol had retardations of about 5 · 5 nm. Since the MA isolated in MTM + EGTA had retardations at least as high as those in hexylene glycol (the rough indicator of the amount of birefringence in vivo), we conclude that MA isolated in MTM + EGTA have stable birefringence of about the level of MA in vivo, and not at the low plateau level of MA isolated in hexylene glycol.

Before turning to other characteristics of MA isolated in MTM + EGTA we mention 2 points related to birefringence measurements which may be of interest. First, MA isolated in hexylene glycol at pH 6-4 had less birefringence than those isolated at pH 6 · 0; after storage, those isolated at pH 6 · 4 had zero retardation and those isolated at pH 6-0 had a stable plateau level of about 50% of the initial retardation, both findings being in agreement with earlier observations on isolations in different pH hexylene glycol solutions (Goldman & Rebhun, 1969). Second, the retardations of MA transferred to hexylene glycol (pH 6 · 0) after being initially isolated in MTM + EGTA decayed at a slower rate than did the retardations of MA isolated initially in hexylene glycol, illustrating another difference between the products of the 2 isolation procedures.

MA were isolated in MTM + EGTA, were stored for 13 days at room temperature, and were then fixed and processed for electron microscopy. As seen in sections, many microtubules were present both in the spindle and aster regions (Figs. 4 — 6). The microtubules were not surrounded by a non-staining (‘clear’) space, as they are in many cells; rather, the microtubules appeared closely associated with darkly stained, amorphous material which sometimes seemed to coat the microtubules and give them a ‘negatively stained’ appearance (Fig. 6), similar to axoneme microtubules seen by Warner & Satir (1973) after thiourea treatment. Also, the microtubules often seemed to be quite close together (Fig. 5). Both the close association of microtubules with amorphous material and the close association of microtubules with each other have been seen in other cells treated with 50% glycerol (Forer & Behnke, 1972), so perhaps the appearances of microtubules in the isolated MA are due to the glycerol in the isolation medium. Or perhaps the adhering material is related to that seen by Goldman & Rebhun (1969) and Rebhun et al. (1974).

In asters of 2 different metaphase MA up to 870 microtubules were seen in single sections (in areas of 20 μm²). In different sections there were between 20 and 100 microtubules per square micron, as in Table 1. The microtubules were between 19 and 22 nm in diameter, and thus microtubules occupied about 0 · 7 — 3 · 0% of the areas counted. The total number of microtubules seen per aster, then, is about the same as seen in asters of MA isolated from Arbacia zygotes (Cohen & Rebhun (1970) counted between 115 and 339 microtubules in different sections of asters), and the micro-tubules seen in our isolated MA occupy about the same relative areas of the sections as do those seen in S. purpuratus zygote MA studied in situ and studied after isolation (review, see table 4 in Forer, 1969). Therefore we presume that the stored MA have not lost many (if any) microtubules, and that the MA microtubules are stable in MTM + EGTA for at least 13 days. Thus, whereas in MA isolated using hexylene glycol microtubules disappear within a few hours (at room temperature) or a few days (at 0° C) (Kane & Forer, 1965; Goldman & Rebhun, 1969), in MA isolated using MTM + EGTA microtubules are still present in apparently normal numbers after storage for 13 days (at room temperature).

The solubility of MA in 0 · 5 M KC1 was studied by observing individual MA (using either phase-contrast or polarization microscopy) as they were treated with 0 · 5 M KC1. The 0 · 5 M KC1 was placed on a slide at the edge of the coverslip, and because lens paper was placed at the opposite edge of the coverslip the KC1 flowed between slide and coverslip past the MA which were being observed.

As observed using phase-contrast microscopy, MA freshly isolated in MTM + EGTA lost the fibrous appearance of the spindle fibres very quickly once 0 · 5 M KC1 reached them; the chromosomes disappeared as well, but a ‘pad’ of cytoplasm usually remained (Fig. 3). The cytoplasmic granules which remained in the remnant cytoplasmic ‘pad’ moved rapidly and apparently freely, whereas previously, in untreated MA, the cytoplasmic granules were stationary. This indicates that material was extracted from the MA. The MA remained soluble in this way during storage for more than 1 week, at room temperature, the only notable change being an increase in time for the spindle fibres to disappear as MA were stored. After about 10 – 14 days, 0 · 5 M KC1 still caused loss of most of the fibres, but slight remnants of fibres seemed to remain, as well as hints of chromosomes.

As observed using polarizing microscopy, the spindle birefringence quickly disappeared (leaving zero detectable retardation) when 0 · 5 M KC1 reached MA freshly isolated in MTM + EGTA (Fig. 7). The MA remained soluble in this way for 8 – 10 days, after which time a very small amount of birefringence remained after addition of 0 · 5 M KC1, an amount which was detectable but too small to measure. The addition of 0 · 5 M KC1 to MA stored for 3 – 4 weeks still caused most of the birefringence to disappear, with only a small amount remaining. The only detectable change from the fresher MA was that as the time of storage increased, more and more residual birefringence remained: about 0 · 5 nm retardation remained after addition of 0 · 5 M KC1 to MA isolated in and stored in MTM + EGTA for 4 weeks, at room temperature. Storage at o °C did not alter the changes described above.

MA isolated in hexylene glycol were initially either (1) completely soluble in 0 · 5 M KC1, except for the asters (MA isolated at pH 6 · 4), or (2) the birefringence disappeared after treatment with 0 · 5 M KC1, leaving a pad of isotropic cytoplasm (MA isolated at pH 6 · 0). After storage for 8 – 10 h at room temperature the MA isolated at pH 6 · 4 had no birefringence, while those isolated at pH 6-o had about 50% of the initial birefringence; 0 · 5 M KC1 seemed to have no effect on such stored MA isolated at either pH.

Thus, while MA isolated with hexylene glycol rapidly lose their solubility in 0 · 5 M KC1, MA isolated in MTM + EGTA are stable (with respect to solubility in 0 · 5 M KG) for 8 – 10 days at room temperature, with most of the birefringence remaining soluble for up to 2 · 5 weeks thereafter.

In one experiment we tested the effects of low temperature on MA isolated in MTM + EGTA, and found that these MA are cold-labile when transferred to one-quarter strength MTM + EGTA: the birefringence disappeared when MA were incubated at 4 °C overnight. This lability was maintained for at least several days after isolation. Thus, like MA in vivo, MA isolated in MTM + EGTA (and MA isolated in tubulin-polymerizing medium (Rebhun et al. 1974)) seem to be cold labile.

MA isolated in hexylene glycol (pH 6 · 0) were transferred to MTM + EGTA immediately upon isolation. While the birefringence of the MA was preserved upon storage, the solubility in 0 · 5 M KC1 decayed nonetheless: for example, after several days in MTM + EGTA 20% of the birefringence remained after addition of 0 · 5 MKCI. As storage time increased, the amount of non-dissolved birefringence increased. This is another difference between MA isolated in 2 different media; something in the MA seems to be altered in the hexylene glycol such that the MA solubility properties are not stable in MTM + EGTA.

We have described a new method for isolating Strongylocentrotus purpuratus zygote MA, based upon the microtubule medium (MTM) originally reported by Filner & Behnkc (1973. 1974) for isolating brain microtubules. In contrast to MA isolated using hexylene glycol, MA isolated using MTM + EGTA have stable birefringence, stable microtubules, and reasonably stable solubility properties, as summarized in Table 2. MA isolated using hexylene glycol lose around 90% of the material found in MA in vivo; while we have no quantitative data on MA isolated in MTM + EGTA, MA isolated in MTM + EGTA seem to have more mass than those isolated in hexylene glycol. Our reasoning for this interpretation is as follows. Visual contrast is achieved in phase-contrast microscopy by converting differences in optical path to differences in amplitude (e.g. Ross, 1967). For identical path lengths, differences in optical path arise from differences in refractive index. Since the contrast between chromosomes and spindle is different for MA isolated in MTM + EGTA than for MA isolated in hexylene glycol, we presume that the spindle refractive indices in MA isolated in MTM + EGTA are closer to those of the chromosomes than are the spindle refractive indices in MA isolated in hexylene glycol. (A closer refractive index would result in less contrast.) Thus, since refractive index is a direct function of concentration of dry matter (Hale, 1958; Ross, 1967), we presume that the MA isolated in MTM + EGTA contain more dry matter than those isolated in hexylene glycol.

The above points seem to be advantages of the MTM + EGTA isolation method warranting further study of these MA. MA isolated in hexylene glycol and then transferred to 2 M sucrose for 12 days have stable birefringence and retain some of their microtubules (Goldman & Rebhun, 1969), but solubility properties were not described (Goldman & Rebhun, 1969). We found that MA isolated in hexylene glycol and subsequently transferred to MTM + EGTA had stable birefringence, but their solubility properties changed within a day. Thus, transfer to stabilizing medium after initial isolation in hexylene glycol would seem to be less satisfactory than direct isolation in a stabilizing medium such as MTM + EGTA. The main drawbacks to isolating MA in MTM + EGTA are those described above, namely, the difficulty in consistently obtaining lysis of zygotes and the variable presence of large clumps of cytoplasm in the preparations of isolated MA.

We wish to emphasize that our object in isolating MA is to get chromosome movement in vitro, and to study the chemistry of the functional motile apparatus. Recently, Cande et al. (1974) successfully obtained chromosome movement in cell models from rat kangaroo fibroblasts; the models were obtained by lysing cells using Carbowax, EGTA, guanosine triphosphate, and Triton-X (at pH 6 · 9), and chromosome movement occurred when anaphase cells were lysed in the presence of 2 · 5 mM ATP. This is an important step towards studying chromosome movement in vitro and in studying the mechanisms of chromosome movement. We hope that the MTM + EGTA method for isolating sea-urchin zygote MA which have stable birefringence, stable micro tubules, and stable solubility properties may eventually lead to chromosome movement in vitro using mass isolation techniques.

This work was supported by grants from the National Research Council of Canada. We thank Drs P. Filner and O. Behnke for sharing unpublished information.

Phase-contrast micrographs were taken using a 40X, NA 0 · 6 Zeiss Jena objective. Polarization-microscope micrographs were taken using a strain-free 63X, NA 0 · 85 objective, at condenser NA = 0 · 5.