Published January 1, 1962
THE MITOTIC APPARATUS:
I S O L A T I O N BY C O N T R O L L E D pH
R. E. K A N E ,
Ph.D.
From the Department of Cytology, Dartmouth Medical School, Hanover, New Hampshire, and the
Marine Biological Laboratory, Woods Hole, Massachusetts
ABSTRACT
INTRODUCTION
Investigations on isolated cell components such
as mitochondria and ribosomes have contributed
much to our knowledge of cell physiology and a
similar approach should be of value with regard
to the problems of cell division. If the mitotic
apparatus (MA) could be isolated in such condition that it would function in vitro, many of the
most perplexing problems of cell division could be
attacked directly. As yet, however, the isolation
process itself presents a considerable problem due
to the apparent lability of the mitotic apparatus.
Since the apparatus does not survive the breakdown of the cell under most conditions, all
attempts at isolation have been based on the
assumption that some form of stabilization would
be required.
T h e mass isolation of the mitotic apparatus
was first achieved by Mazia and D a n (1), using
echinoderm eggs as experimental material. G r a m
quantities of such eggs can be obtained from a
single animal and are extremely synchronous for
several divisions after fertilization, thus making
available to the investigator large numbers of
cells in any desired stage of division. In the experi
ments of Mazia and Dan, extended treatment o
metaphase eggs in 30 per cent ethanol at - 1 0 ° C
was used to stabilize the mitotic apparatus. T h e
eggs were then transferred to a dispersing agent,
usually digitonin (2), to break the cells and liberate
the MA. The possibilities for chemical studies of
the M A attendant upon this isolation have been
exploited by M a z i a and his collaborators in a
number of investigations (3-6).
Although these experiments were of considerable
significance in demonstrating that a structure
closely approaching the classical mitotic figure
could be isolated intact from dividing cells, there
is some question of the possible modification of
the native structure of the M A by the extended
ethanol treatment involved in this isolation. This
problem was recognized by Mazia and led to the
development of the dithiodiglycol ( D T D G )
method of isolation. Previous work by Mazia
and his collaborators on the alcohol-digitonin M A
had suggested that sulfur bonds might be involved
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The isolation of the mitotic apparatus (MA) from the echinoderm egg was studied in de
tail, with particular attention given to the factors governing its stability. Successful isolation
depends mainly on the p H of the isolation solution, slightly acid values being required.
The use of a 1 M solution of hexanediol, buffered at p H 6.0 to 6.4, gives high yields of stable
MA, while M A of poorer quality can be isolated in water buffered at p H 5.5 to 5.8. Isolation is possible only over a very narrow range of pH, as the cells become more difficult
to break at lower values and the M A becomes unstable at higher values. Within this range
the fibrous structure of the M A varies with the pH. T h e isolated M A disintegrates slowly
when transferred to water at p H 7 and dissolves rapidly in solutions of high ionic strength.
Published January 1, 1962
48
mitotic apparatus directly from the living cell
u n d e r mild conditions, is presented here.
MATERIALS
AND METHODS
Gametes
The eggs of the sea urchins Arbacia punctulata and
Strongylocentrotus purpuratus were used as experimental
material. The gametes of A. punctulata were obtained
by the application of 10 volts of alternating current
across the test, using lead electrodes (15). The
gametes of S. purpuratus were obtained by the injection of 1 ml of 0.53 M KCI into the body cavity. The
eggs were shed into 100 to 200 ml of sea water and
washed in additional sea water before use. Sperm
were diluted in the ratio of 1 drop of " d r y , sperm to
25 ml of sea water. A sample of eggs from each female
was tested for fertilizability and those showing less
than 95 per cent were rejected.
Removal of the Fertilization M e m b r a n e
The liberation of the mitotic apparatus from the cell
requires that the eggs be broken by mechanical,
osmotic, or other means. This can be accomplished
easily only if the eggs lack the tough fertilization
membrane. In these experiments the membrane was
removed by treating the eggs with urea immediately
after fertilization.
Moore (16) first showed that the treatment of unfertilized echinoderm eggs with 1 M urea prevented
the formation of a membrane at fertilization. In later
experiments it was shown that other non-electrolytes
were equally effective and that the effect was counteracted by the addition of salts to the non-electrolyte
solutions (17, 18). Moore noted also that the membrane of the eggs of Dendraster excentricus could be dissolved by urea treatment immediately after fertilization (19), and Chase (20) extended this observation
to Strongylocentrotus purpuratus. The membrane becomes more resistant several minutes after fertilization and is no longer affected by urea.
The fertilization membrane of A. punctulata can be
removed by urea treatment if the eggs are transferred to urea immediately after fertilization, and a
sufficient dilution of the sea water salts is achieved.
The following procedure has proven successful with
these eggs. One ml of a concentrated egg suspension is
stirred into 10 ml of sea water contained in a 12 ml
centrifuge tube. Several drops of dilute sperm are
added and timing begun. The tube is stirred for 15
seconds, spun in a hand centrifuge to sediment the
eggs, and the sea water poured off. Ten ml of 1 M
urea, previously adjusted to pH 7.5 with K O H , is
added and the eggs dispersed into this solution. The
tube is immediately spun again, the process repeated,
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in its structure (2). I n addition, the sulfhydryl
c o m p o u n d , mercaptoethanol, had been shown to
have a marked effect on division (7). These
observations led to the development of an isolation
method based on the use of an excess of dithiodiglycol (the oxidized form of mercaptoethanol) as
a stabilizing agent. A n u m b e r of different procedures using dithiodiglycol have been published
(8-10), but all involve the transfer of living metaphase eggs to a solution containing 0.15 M dithiodiglycol as its essential ingredient. After penetration
of the D T D G the cells are broken by mechanical
means, liberating the mitotic apparatus. This
isolation procedure overcomes m a n y of the objections to the ethanol procedure, since the M A is
liberated directly from the living cell without
pretreatment. However, the role of the dithiodiglycol in this isolation procedure is not clear, its
use being justified largely on pragmatic grounds.
I n the studies reported here the isolation of the
mitotic apparatus was investigated in some detail,
with particular attention given to the factors
governing its stability. Information gained from
these studies was then used to develop an isolation
procedure involving a m i n i m u m of chemical
treatment, with the aim of producing a m i n i m u m
modification of the MA. Proper evaluation of any
procedure for the isolation of cytoplasmic organelles requires, of course, the study of their fine
structure with the electron microscope. This presents a p r o b l e m in the case of the mitotic apparatus, since there is no " s t a n d a r d " intracellular
unit to which the isolates can be compared.
Electron microscopic investigations of dividing
echinoderm eggs have not as yet given unequivocal
evidence as to the structure of the intracellular
mitotic apparatus. Kurosumi (11) observed a
rather coarse fibrous structure, while Gross et al.
(12) found no evidence of fibrous organization in
the spindle region of conventionally fixed cells.
This problem may be due in part to the difficulties
of obtaining adequate fixation of marine material,
since electron micrographs of dividing m a m m a l i a n
cells show the presence of m a n y delicate fibers
in the spindle region (13). Preliminary electron
microscopic studies of the mitotic apparatus
isolated by the procedure to be described here
show considerable fibrous organization, m a n y fine
tubules with a d i a m e t e r of the order of 200 A being
present (14). A detailed description of this isolation procedure, which allows isolation of the
Published January 1, 1962
RESULTS
Original Observations
These experiments were begun using the dithiodiglycol method of M a z i a in its original form
(8); later modifications have since been pub-
lished (9, 10). This method consists in the transfer
of metaphase eggs to a solution containing 0.15 M
D T D G , 1 M glucose and 6 X 10-~M ethylenediamine tetraacetic acid (EDTA), p H 6.2 to 6.4. The
D T D G penetrates the eggs rapidly and the solution is then shaken to break the eggs and liberate
the mitotic apparatus. Some free mitotic apparatuses were produced by this method, but yields
were small and difficulty was experienced in
obtaining complete dispersal of the cells without
d a m a g i n g the mitotic apparatus. Cytolysis of the
eggs in penetrating non-electrolytes was investigated as a possible method of obtaining better cell
breakdown. T h e substitution of molar ethylene
glycol for glucose in this mixture improved the
results. After several minutes the eggs swelled to
several times their normal size and moderate agitation was sufficient to lyse them, liberating und a m a g e d M A into solution. Reduction in the
a m o u n t of D T D G present in the solution had little
effect and mitotic apparatuses were sometimes
observed after cytolysis of the eggs in solutions
containing only ethylene glycol.
The E D T A was also eliminated from the mixture and the concentration of divalent ions reduced
by washing the eggs in isotonic monovalent salt
solution before cytolysis. Since the eggs are unstable in pure sodium or potassium chloride, the
19:1 mixture of isotonic sodium and potassium
chlorides r e c o m m e n d e d by C h a m b e r s (21) for
membraneless eggs was used. T h e metaphase
eggs are passed through two changes of this solution before cytolysis. In addition to reducing the
concentration of divalent ions below effective
levels, this procedure also removes the bicarbonate present in the calcium-free sea water and
allows better p H control during the subsequent
procedures.
Use of Longer Chain Glycols
The fact that isolation of the mitotic apparatus could sometimes be obtained in solutions
containing only ethylene glycol showed that
isolation without stabilization was possible u n d e r
suitable conditions. Isolation was never observed
in solutions containing only glucose or other nonpenetrating compounds, indicating that cytolysis
of the egg might be necessary for successful isolation. T h e substitution of propylene for ethylene
glycol gave slightly better results, suggesting that
the rate of penetration might be a factor and that
R. E. KANE Mitotic Apparatus: Isolation by Controlled pH
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and fresh urea added. This second change of urea is
allowed to remain on the eggs until approximately
21/~ minutes after fertilization. The eggs are then
spun down gently, the urea decanted, and calciumfree sea water containing bicarbonate, p H 7.8, is
added. This is repeated and fresh calcium-free sea water added. The eggs are transferred to large (130 mm)
Stender dishes and allowed to develop on the sea
table at 19 to 21°C. The dilution of the sea water
salts in the final urea solution is thus approximately
100 times. Higher ratios of eggs to urea often result
in the presence of residual membranes. The membranes of S. purpuratus are more easily removed and
require a dilution of only ten times. In this case only
one change of urea, pH 6.5, is used, the urea being
left on until 21/~ minutes after fertilization. These
eggs become quite fragile and frequently cytolyze in
urea at pH values above neutrality, but are stable at
the lower p H used. The eggs of & purpuratus are
grown in a constant temperature bath at 16°C. The
calcium-free sea water used in both cases reduces the
hyaline layer and lessens the tendency of the eggs to
clump, but seems to have no effect on cleavage. There
is some variation in the response to urea with both
species over the course of the breeding season.
Four tubes can be handled easily, allowing the
treatment of 4 ml of eggs. The purpose of the experiments described here was generally to test a large
number of different isolation procedures, rather than
to prepare massive batches of isolated mitotic apparatuses. Two ml of eggs was sufficient for most experiments, as it allowed four isolation procedures to be
run at metaphase. Since the different isolation procedures were compared on the basis of the appearance of the resulting MA, and minor differences in
procedure often produce considerable differences in
the visible structure, an internal standard was required. Such a standard was obtained through a
control isolation run in parallel with the experimental
procedures. The control was an isolation method
which had previously proven successful, and which
was carried out in an identical fashion in each experiment. If the control procedure produced MA identical to those in previous runs, then any differences in
the structure of the MA in the experimental procedures could be considered with confidence to be
due to the procedure itself.
All observations on the isolated mitotic apparatus
were made with phase contrast microscopy.
Published January 1, 1962
p H Effects on Hexanediol Isolation
Isolated mitotic apparatuses are often observed
after lysis of metaphase eggs in a solution containing only 1 M hexanediol, but control of the
p H of the solution is required to obtain complete
reproducibility in the appearance and yield of
MA. Since other experiments had shown that it
was desirable to hold the concentrations of all
cations in the solution to as low a value as possible,
m i n i m u m amounts of buffer are used. Experiments
at p H 6.0 and below are buffered with 0.005 M
potassium phthalate, and 0.01 M potassium phosphate is used from p H 6.0 to 8.0. Tests at p H 6.0
show these buffers to be indistinguishable with
regard to effects on isolation. A series of isolations
in 1 M hexanediol over a range of p H demonstrates that the stability of the mitotic apparatus
depends directly on this variable. No M A can be
found after lysis at p H 8.0. At p H 7.5 some mitotic
apparatuses can be found in the lysate, but they
50
are rapidly fragmenting and disappear from the
solution after a short time. At pH values below
7.0, stable mitotic apparatuses are isolated. In the
range of p H from 7.0 to 6.5 the mitotic apparatuses are small and irregular and have little
evident structure, as shown in Fig. 1. The most
satisfactory range for isolation is from pH 6.4 to
6.0, the apparent fibrous structure of the M A
increasing with decreasing p H in this range. At
p H 6.0 the structure of the isolated mitotic apparatus as seen in phase contrast (Fig. 2) approaches
that of the classical mitotic figure familiar from
fixed and stained cells. It should be emphasized,
FIOUEE 1
Mitotic apparatus isolated in pH 6.8, 1 M hexanediol, in same solution. Asters small and without
visible structure, spindle fibers poorly defined.
Phase contrast, X 600.
however, that this structure is almost entirely a
phase image, the M A being essentially invisible
in bright field. Below p H 6.0 the cells become
more difficult to break, the suspensions become
coarser, and the mitotic apparatuses are often
contaminated with yolk granules.
Isolation in Water
One of the more interesting problems connected with this isolation method is the role of
the hexanediol. Although no definite evidence
bearing on this problem has yet been obtained,
the possibility of a chemical "stabilizing" function
for hexanediol was rendered unlikely by the
demonstration of its dispensability. Isolation of
the M A in distilled water was occasionally
observed in the early experiments, but the result
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more rapidly penetrating glycols might be more
effective. The six-carbon hexanediols caused
extremely rapid cytolysis of the cells and gave
excellent dispersion of the cytoplasmic particulates. The 1,6 and 2,5 compounds were equally
effective, the latter being chosen for most experiments as it is a liquid and more easily handled.
Eggs transferred to 1 M 2,5 hexanediol swell very
rapidly to several times their normal volume and
cytolyze, the entire process requiring only about
15 seconds. In the case of A. punctulata the pigment
is extracted from the cytolyzed eggs and, when
centrifuged gently, the cells pack to a white mass,
leaving a dark red supernatant. This elimination
of the pigment proved very convenient and a
change of hexanediol solution after cytolysis was
included in experiments on eggs of both species.
The cytolysis procedure is carried out as follows:
the metaphase eggs are spun out of the second
change of monovalent salt solution, the supernatant decanted, and a volume of 1 u 2,5 hexanediol solution equal to ten times the volume of
packed ceils added with gentle stirring. After 30
seconds to 1 minute the eggs are sedimented
again, the supernatant decanted, and fresh
hexanediol solution added. The cells are then
dispersed by very gentle swirling of the tube,
giving a fine suspension of the cytoplasmic particulates and, under proper conditions of pH, high
yields of mitotic apparatuses.
Published January 1, 1962
was not reproducible. T h e p r o n o u n c e d influence
of p H on the hexanediol isolation immediately
suggested t h a t a similar effect m i g h t be involved
here. This is the case, proper p H control allowing
isolation of the mitotic a p p a r a t u s in water. After
lysis at p H 6.0 in w a t e r buffered with 0.005 M
phthalate, mitotic apparatuses are only rarely
seen. As the p H is reduced the cells become more
difficult to break, b u t more mitotic apparatuses
a p p e a r in the lysates. A t p H 5.7 to 5.8 the
mitotic apparatuses are small a n d transparent, b u t
at p H 5.5 to 5.6 stable mitotic apparatuses, simi
lar in a p p e a r a n c e to those isolated at p H 6 in
has been r u n several h u n d r e d times on the eggs of
b o t h species with completely reproducible results.
M a n y variations in this procedure have been
tested and all have yielded poorer results. Reduction in the c o n c e n t r a t i o n of hexanediol requires
the use of lower p H values a n d gives coarser
suspensions. Increased salt concentration also
has deleterious effects. S o d i u m a n d potassium
h a v e little effect until a concentration of 0.05 to
0.1 M is reached; in this range the eggs become
more difficult to break a n d the mitotic a p p a r a tuses are c o n t a m i n a t e d with yolk granules. T h e
addition of calcium or m a g n e s i u m also causes
the eggs to become more resistant to lysis, these
FIGURE
Mitotic apparatuses isolated in pH 6.0, l M hcxanediol, in same solution. Pronounced fibrous
structure evident. Phasc contrast, X 600.
hexanediol, are isolated (Fig. 3). This m e t h o d is
m u c h less satisfactory t h a n the hexancdiol procedure for the isolation of the M A , as the cells disperse into a r a t h e r coarse suspension and the
mitotic apparatuses are usually surrounded by
yolk particles, b u t it clearly demonstrates that
acid p H alone is a sufficient stabilizing agent.
This p H is very close to the limit at which any
dispersion of the cells can be obtained. Below p H
5.4 the cells merely f r a g m e n t into a few large
clumps which c a n n o t be dispersed further, making
isolation impossible.
Other Observations on Isolation
T h e p H 6.0 to 6.4, 1 ~ hexanediol procedure
is the most generally useful isolation m e t h o d a n d
ions being effective at 10 -~ M. C o n t r a r y to w h a t
one m i g h t expect, the addition of the chelating
agent E D T A at 10-3M does not aid in cell lysis
b u t instead causes more yolk c o n t a m i n a t i o n of
the MA.
It has not been possible to isolate the mitotic
a p p a r a t u s in solutions of n o n - p e n e t r a t i n g nonelectrolytes, such as glucose and glycerol, at
p H 6. T h e cells must be broken by mechanical
means in such solutions, a n d mitotic apparatuses
are usually not detectable after breakage. A few
rapidly disintegrating asters are sometimes seen,
b u t these soon disappear. A 1 M solution of glycerol
buffered at p H 5.5 is m u c h inferior to w a t e r at
the same p H as a n isolation medium, the M A in
glycerol being completely structureless a n d
covered with yolk.
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FIGURE
Mitotic apparatus isolated in p H 5.5 water, in
same solution. Structure similar to that of mitotic
apparatuses isolated in hexancdiol at pH 6.0.
Phase contrast, X 600.
Published January 1, 1962
Action of Dithiodiglyeol
I t is of interest to re-examine the dithiodiglycol m e t h o d in the light of these studies. T h e
most recent modification of this m e t h o d (10) involves the use of a 0.15M solution of this comp o u n d without added glucose. T o c o m p a r e the
effects of D T D G a n d hexanediol, a 0.15 M solution
of each c o m p o u n d was used, both buffered at
p H 6 with 0.005 M phthalate. Mitotic apparatuses
were found in both solutions after lysis a n d h a d
similar morphology, If the experiment is repeated
at p H 7, no mitotic apparatuses are found in
centrifugation and resuspension, a pellet composed entirely of M A is obtained.
T h e mitotic a p p a r a t u s is stable indefinitely in
the p H 6, I ~ hexanediol solution used for isolation, and retains its fibrous structure u n d e r these
conditions. I n contrast to the effects of higher p H
during isolation, the isolated M A is stable in
hexanediol solutions above neutrality. I n hexanediol solution at p H 7 the visible fibrous structure is reduced, while at p H 8 the M A is practically structureless (Fig. 4).
T h e isolated M A is also stable in water buffered
at p H 6.0 to 6.4. Such a solution has been used
either solution. This suggests t h a t hexanediol a n d
dithiodiglycol are equivalent a n d t h a t the action
of D T D G is due to its rapid penetration and
subsequent effect on cell lysis r a t h e r t h a n to the
disulfide group.
Properties of the Isolated Mitotic Apparatus
T h e initial cytolysis of the eggs is carried out
at the same t e m p e r a t u r e at which the eggs are
grown. As soon as the eggs are lysed and the
mitotic a p p a r a t u s liberated, the samples are
placed in ice a n d kept at this t e m p e r a t u r e d u r i n g
all subsequent manipulations.
T h e mitotic
apparatuses are separated from the small particulate components in the lysate by differential
centrifugation. Two minutes at 500 g is sufficient
to sediment t h e m and, after several cycles of
52
]~IGURE 5
Mitotic apparatus isolated in pH 5.5 water, immediately after transfer to water, pH 7.0. M A
appears granular, spindle region breaking down.
Phase contrast, X 600.
for washing a n d for short term storage of the
mitotic apparatuses, as they retain their solubility
properties for some days in this solution. If freshly
isolated mitotic apparatuses are transferred to
water buffered at p H 7.0 with 0.01 ~ potassium
barbital, they swell, lose structure, a n d begin to
disintegrate. As illustrated in Figs. 5 a n d 6, the
disintegration begins in the spindle region, often
freeing the chromosomes from the MA. I t is of
interest to note that the chromosomes do not
immediately disperse, but r e m a i n together in
their metaphase a r r a n g e m e n t for some time after
release from the MA. After a few hours in this
solution only the chromosomes and small fragm e n t i n g asters r e m a i n (Fig. 7) and, eventually
even the asters disintegrate completely. T h e
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FIGURE 4
Mitotic apparatus isolated in pH 6.0, 1 M hexanediol, transferred to pH 8.0, 1 M hexanediol after
isolation. Fibrous structure no longer evident.
Phase contrast, X 600.
Published January 1, 1962
response of the M A is similar, b u t slower, in
water buffered with 0.01 M potassium phosphate
or Tris, complete disintegration of the M A often
requiring u p to 1 day. This property of disintegrating in water at p H 7 is a sensitive indicator
of the condition of the M A , as it is lost very rapidly
after isolation. Disintegration is similar in 1 M
glycerol buffered at p H 7 with 0.01 barbital.
T h e process is slower in 1 M ethylene glycol at
p H 7 a n d requires several days in 1 M propylene
glycol at p H 7.
T h e integrity of the mitotic a p p a r a t u s is retained
in water buffered at p H 7 containing 10-3M cal-
dissolved. T h e rapid dissolution makes it difficult
to d e t e r m i n e w h e t h e r there is specific action on
any particular region of the MA, a l t h o u g h there
is some evidence indicating t h a t the spindle region
is attacked first. Solubility in solutions of high
ionic strength is retained for a considerable
period, M A stored in w a t e r at p H 6.4 responding
to these salt solutions for several days after isolation. Previous exposure of the M A to 10 -3 M magnesium chloride does not affect this response.
cium or m a g n e s i u m chloride. T h e mitotic a p p a ratuses lose m u c h of their a p p a r e n t fibrous structure in these solutions (Fig. 8), b u t are stable
indefinitely. T h e M A is also stable in 0.1 to
0.2 M KC1 buffered at p H 7.0 and has a similar
appearance.
T h e mitotic a p p a r a t u s dissolves very rapidly
in solutions of higher ionic strength, such as
0.5M KC1 buffered with 0.01 M phosphate at
p H 7 or 0.1 ~ sodium pyrophosphate adjusted to
p H 7. T h e process is so rapid t h a t it can be
observed only by the addition of the salt solution
directly to the mitotic a p p a r a t u s on a microscope
slide. Fig. 9 illustrates the a p p e a r a n c e of the M A
immediately after the addition of the salt solution;
a few seconds later the M A h a d completely
FIGURE 7
Mitotic apparatuses isolated in pH 6.0, 1 M
hexanediol, transferred to water, pH 7.0, after
isolation. Aftcr 30 minutes in water. Thrce small,
fragmenting asters and one chromosome set visible.
Phase contrast, X 600.
DISCUSSION
Isolation of the mitotic a p p a r a t u s in w a t e r at
p H 5.5 to 5.6 is a simple procedure a n d it is somew h a t surprising t h a t it has not been observed
previously. T h e fact t h a t the classical mitotic
figure of light microscopy is obtained after acid
fixation indicates t h a t p H has a p r o n o u n c e d
effect on the M A , a n d the experiments of M. R.
Lewis (22) show t h a t even living cells will reversibly form visible spindle fibers on acid treatment. O n e of the difficulties t h a t m a y h a v e prev e n t e d such isolation is the fact t h a t isolation is
possible, at least in the case of the u r c h i n egg,
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FIGURE 6
Same M A as Fig. 5, several minutes later. Chromosomes are now free of M A and are seen in polar
view. Spindle dissolved, asters still intact. Phase
contrast, X 600.
Published January 1, 1962
only over a very narrow range of pH. T h e mitotic
a p p a r a t u s is unstable at p H values above this
range, while the cells are difficult to disperse and
the M A c o n t a m i n a t e d with yolk to the point of
invisibility at more acid values. This difficulty
i n dispersing the cytoplasm at lower p H values
m a y be due to the fact t h a t such values a p p r o a c h
the isoelectric point of the cell proteins; essentially
all of the protein present in extracts of unfertilized
or fertilized eggs is precipitated at p H 4.5 (23).
Isolation in water, although of considerable
theoretical interest, is unsatisfactory for the
routine p r e p a r a t i o n of the mitotic apparatus. T h e
the glycols are dispensable both d u r i n g a n d after
isolation by lowering the pH, it seems unlikely
t h a t they have some u n i q u e "stabilizing" action
on the MA.
T h e evaluation of an isolation m e t h o d requires
the comparison of the native a n d isolated structure by a variety of methods. T h e most direct
method, of course, is t h r o u g h the comparison of
the isolated M A with that of the living cell by
means of phase contrast, interference, or birefringence methods. T h e usefulness of these methods
is limited with regard to the u r c h i n eggs used
here, as the yolk granules present in the cytoplasm
FIGURE 9
Mitotic apparatuses isolated in pH 6.0 1 M hexanediol, immediately after irrigation with 0.1 M
sodium pyrophosphate. MA in process of breaking
down. Phase contrast, X 600.
use of a 1 M solution of hexanediol allows isolation
to be m a d e at p H 6.0 to 6.4, a range in which the
cytoplasm is easily dispersed a n d the mitotic
apparatuses are essentially free of yolk c o n t a m i n a tion. T h e m e c h a n i s m by which hexanediol acts
to raise the p H at which isolation is possible is not
known, a l t h o u g h a similar effect is seen after
isolation, the M A being stable at higher p H values
in hexanediol solutions t h a n in water. I n b o t h of
these cases there appears to be some relation
between the chain length of the glycols a n d their
effectiveness. T h e longer chain length glycols are
more effective in raising the p H at which the M A
c a n be isolated a n d also in preserving the isolated
M A at values above neutrality. However, since
make such studies difficult. I m p r o v e d images c a n
be obtained t h r o u g h the use of flattened or
centrifuged eggs, but with the a t t e n d a n t risk of
thereby inducing structural modifications. T h e
only satisfactory solution to this p r o b l e m lies in
the use of relatively t r a n s p a r e n t cells. C o m p a r i s o n
at the fine structural level, which has proven so
valuable with other organelles, does not yield
unequivocal answers in the case of the mitotic
a p p a r a t u s because of the difficulty of d e t e r m i n i n g
the ultrastructure of the intracellular MA. T e m porarily then, we must evaluate the isolation on
the basis of indirect evidence. T h e m e t h o d presented here is based on osmotic lysis in w a t e r or
hexanediol solution u n d e r slightly acid conditions.
54
ThE JOURNAL OF CELL BIOLOGY • VOLUME 1~, 196~
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l~GURE 8
Mitotic apparatuses isolated in pH 6.0, 1 u hexanediol, transferred to watcr, pLI 7.0, containing l 0 -a M
MgCl~. Asters have little structure, while spindle
region is very bright and without visible structure.
Phase contrast, X 600.
Published January 1, 1962
for chemical studies of the mitotic apparatus. The
M A isolated in 1 u hexanediol at p H 6.4 are
essentially free of yolk and can be freed of soluble
contaminants by repeated washing with water at
p H 6.4. They are easily soluble in solutions of
very low or relatively high ionic strength. The
disintegration of the M A in water buffered at
p H 7 is of particular interest, since it takes place
fairly rapidly in the spindle region and much more
slowly in the asters, suggesting that it may be
possible to prepare separately the water-soluble
aster components and the water-soluble spindle
components. The M A is stable in 0.1 to 0.2 M
KC1, but solubility rises again above 0.3 M KC1.
Considerable amounts of protein go into solution
in both water and 0.5 M KC1, and it will be of
interest to compare the components soluble under
varying salt conditions. These problems are now
under investigation.
This investigation was supported by a PHS research
grant, RG-8626, from the Division of General Medical Sciences, Public Health Service, and also by
grant G-9627 from the National Science Foundation.
Received for publication, July 25, 1961.
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R. E. KANE Mitotic Apparatus: Isolation by Controlled pH
55
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Osmotic lysis appears to be necessary, since
breakage of the eggs in non-penetrating nonelectrolytes does not give successful isolation.
This osmotic shock may have some effects on the
mitotic apparatus, although the isolated M A
shows little response to changed osmotic conditions. The acid p H is also required, since isolation
of stable M A at p H values above neutrality has
so far proven impossible in all media tested. The
slightly acid conditions required for stability
should not have serious effects on the MA, as the
p H values used are only slightly below those estimated for the intracellular pH, which are usually
in the range of p H 6.6 to 6.8 (24, 25). The acid
p H of the isolation media is responsible for the
fibrous appearance of the isolated MA, but this
can be reversed by transfer to hexanediol solutions above neutrality. It is of interest to note in
this connection that the fibrillation of the spindle
of living cells induced by acid in the experiments
of M. R. Lewis (22) also reversed when the cells
were returned to normal conditions. The acid
treatment did not cause permanent damage to
the spindle, as the cells usually completed division.
This isolation method provides suitable material