SPINDLE MICROTUBULES: THERMODYNAMICS OF
IN VZVO ASSEMBLY AND ROLE IN
CHROMOSOME MOVEMENT *
E. D. Salmon
Marine Biological Laboratory
Woods Hole, Massachusetts 02543
and
Department of Biology
University of Pennsylvania
Philadelphia, Pennsylvania 19104
The orderly segregation of the chromosomes during mitosis is accomplished
through the temporary assembly and function of the mitotic spindle. The
major theories of mitotic mechanisms have been, and still are, based primarily
on proposed mechanical and chemical characteristics of the chromosomal and
continuous spindle fibers. Microtubules are the major anisotropic structural
elements of these fibers.'-:{
The following discussion is concerned both with the mechanisms of spindle
microtubule assembly and disassembly, and with the structural and functional
role of microtubules in producing and regulating chromosome movement. In
particular, I shall present the results of experiments in which hydrostatic pressure induced depolymerization of spindle microtubules in living cells. These
pressure studies were initially conducted to test the InouC "dynamic equilibrium" model of spindle assembly, and to examine the possible role ( o r
roles) of microtubule polymerization-depolymerization in producing forces for
chromosome movement.
SPINDLE
MICROTUBULE
ASSEMBLY
Historically, studies with polarization microscopy in which the birefringent
spindle fibers in living cells were reversibly abolished by various agents (principally cold and colchicine) led InouC
to hypothesize that spindle microtubules are in a labile, dynamic equilibrium with a cellular pool of subunits.
Extensive data substantiate the lability of spindle fiber microtubules in vivo:
numerous chemical and physical treatments produce rapid and generally reversible disassembly and assembly."
Initially, temperature studies of the assembly of metaphase-arrested spindles
in vivo provided the major support for InouC's hypothesis. In 1952 InouC
discovered that changes in equilibrium temperature produced predictable, reversible changes in equilibrium birefringence retardation (BR) as measured in
the central half-spindle region of meiosis I spindles in Chaetopterus pergamentaceous oocytes. Equilibrium BR increased as temperature increased, with BR
I(
:>Thiswork was supported by grants CAI0171 and GM20644-01 from the National Institutes of Health, and grant GB31739X from the Natonal Science Foundation.
383
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Annals New York Academy of Sciences
approaching an upper limit at higher temperatures. Assuming that the measured BR ( B ) is directly proportional to the amount of oriented assembled
subunits in that region, that the upper limit of B ( A , , ) is proportional to the
total amount of the orientable material in the same region, and that only the
equilibrium constant between assembled and unassembled material is influenced
by temperature, InouC and Morales
formulated a temperature-dependent
equilibrium constant for the assembly, based on a simple equilibrium between
monomer and polymer:
Gv
and
’
Thermodynamic (van’t Hoff) analysis produced a reasonable, linear relationship between In K and l / T ° K , and yielded large positive values of enthalpy
(AH= 28.4 kcal/mol) and entropy (AS= 101.0 eu) of association. The
standard free energy of polymerization (AGO) was less than -1 kcal/mol at
normal physiological temperature ( ~ 2 0C” ), consistent with the characteristic
lability demonstrated for the spindle fibers. Thermodynamic parameters of
such sign and magnitude are similar to those of the in vitro assembly of actin l o
and tobacco mosaic virus (TMV) protein,l1. 12 which led InouC to suggest that
similar mechanisms govern the assembly of the spindle protein subunit molecules
into the oriented polymers of the spindle fibers. The large positive AH and
AS values of association were explained on the basis of the dissociation or
“melting” of constricted bound water away from the subunits during the association
More recent investigations have substantially supported the assumptions
and assembly hypothesis of InouC. First, a direct relationship between either
microtubule number or tubulin content and spindle fiber BR has been determined. Changes in the magnitude and distribution of spindle BR, whether
normal or experimentally induced, are correlated with changes in the number
and distribution of microtubules.R,zoo-?4 The loss of spindle BR has been shown
to result from depolymerization of the microtubules into small subunits, not
from fragmentation or disorientation of the microtubules.sl 5 1 Microtubules are
essentially parallel in the central spindle region, and the BR can be totally
accounted for theoretically by the aligned microtubules.lO 2 5 Stephens has
also demonstrated that the amount of tubulin extracted from the isolated
mitotic apparatus of marine oocytes is proportional to the central half-spindle
BR in vivo. Therefore, BR is a good measure of either the number of microtubules in the spindle fibers or the concentration of polymerized tubulin in the
spindle region.
Secondly, the existence of a spindle tubulin pool has been substantiated by
the following evidence: spindle assembly is independent of immediate protein
synthesis; 81 28 there is a presynthesized cellular pool of t u b ~ l i n , ’ ~more
- ~ ~ than
sufficient to account for the maximal content of spindle microtubules; 31-33 and
the apparent spindle tubulin pool at metaphase can be controlled experimentally
through changes in environmental temperature during prophase.2s
Finally, the simple equilibrium model accounts for the changes in equilibrium BR induced by changes in temperature in the active metaphase spindles
of the sea urchin Strongylocentrotus droebachiensis,15 the starfish Asterias
Salmon: Assembly Thermodynamics of Spindle Microtubules
385
forbesii,"' and the endosperm of Tilia ai?iericana,I';as well as several meiotic
metaphase-arrested spindles.l7-lYHeavy water enhances the assembly of spindle
microtubules,fi as would be expected for a hydrophobically bonded system.
Cytoplasmic brain microtubules have been polymerized recently in vitro. These
microtubules show the expected reversible depolymerization sensitivity to cold,
:35
colchicine, and dilution of tubulin
If the InouC assembly hypothesis is correct, then it should also predict the
changes in spindle microtubule assembly (as measured by spindle BR) with
increases in hydrostatic pressure, as it does for temperature. In fact, the model
predicts that the degree of microtubule polymerization at normal physiological
temperatures would be substantially reduced by moderate pressures (less than
5,000 psi). The influence of pressure on a reaction is directly related to the
difference in-the magnitude of the molar volumes of the products and the
reactants (AV; le Chatelier's principle). The in vitro endothermic self-assembly
~
3y and flagellin 39 typically involves an
of TMV protein,'?, a ~ t i n , 3myosin,3'*
unusually large positive A V (range 150-700 ml/mol subunit), as well as large
positive values of AH and
The early classical studies of fixed and stained cells, or stabilized spindles,
by Pease I 1 and Zimmerman and Marsland,'?. I 1 demonstrated that pressure
could disorganize the spindle structure and prevent mitotic function. The
degree of microtubule assembly, however, was difficult to assess quantitatively
by the methods employed.44 To avoid such problems, a new miniature,hydrostatic pressure chamber for observing living cells was designed and developed
by Salmon and Ellis.'5 A minimum of peripheral equipment permitted the
generation of pressures up to 10,000 psi, with simultaneous control of temperature. This chamber provided for the first time the opportunity to examine
quantitatively, with polarization microscopy, the effects of pressure on polymerization of spindle microtubules and, with phase contrast microscopy, the
accompanying effects of pressure on the movement of chromosomes.
General Effects of Hydrostatic Pressure
The pronounced effect of pressure on spindle microtubule polymerization
1 (see also FIGURE
8 ) . FIGURE2 shows that after
is illustrated in FIGURE
pressure is applied, the BR of the metaphase-arrested meiosis I spindle in
Chaetopterus pergainentaceous oocytes decreases rapidly, while the spindle
shortens more slowly. Both the BR and spindle length reach equilibrium values
that depend on the magnitude of the pressure applied. When atmospheric
pressure is restored after moderate pressurizations, the spindle BR rapidly
increases while, more slowly, the spindle elongates. If the spindle is depolymerized completely (by pressure greater than 3,500 psi at 22" C ) , there is a
delay after pressure is released before the spindle reappears; this delay increases
to 30 sec following a 9,000 psi pressurization. Once the spindle reappears,
however, it repolymerizes rapidly.
The effect of pressure on active-metaphase spindles is essentially the same
1 and 3 ) , but some
as the effect on metaphase-arrested spindles (FIGURES
variation in recovery may occur. Although a single Chaetopterus spindle
could be repeatedly pressurized and depressurized as many as 15 times with
no irreversible effects (the cells could be fertilized and would develop normally) ,", abnormalities of varying severity did occasionally occur in spindles
386
Annals New
York Academy of Sciences
FIGURE1. Examples of changes in equilibrium metaphase spindle assembly with
increasing magnitude of hydrostatic pressure in the first mitosis of Strongylocentrotirs
droebacliiensis at 8" C, as observed with polarization microscopy. The intensity due
to the birefringence of the chromosomal and continuous fiber microtubules (2.25 nm
at atmospheric pressure) is brighter than the background. The astral rays perpendicular to the spindle interpolar axis subtract from the compensator BR and appear
darker than the background. The eggs have been flattened to remove light-scattering
yolk from above and below the spindle. Pressure of 2,500 psi induces a substantial
decrease in spindle BR (to about 0.75 nm), accompanied by a shortening of the
spindle interpolar distance to equilibrium values. At 4,000 psi the spindle becomes
completely depolymerized within 5 min., but the original spindle region remains generally free of yolk granules. Bar= 10 pm.
of S. droebachiensis, depending o n the length of the mitotic stage, the length
and magnitude of pressurization, and the apparent chromosome bridging in
anaphase induced by pressure. Pressures that produce significant spindle depolymerization d o not greatly alter the timing of the mitotic cycle unless they
are held for a prolonged period (FIGURE3 ) . Spindles appear susceptible to
complete depolymerization by pressures between 3,000 and 7,000 psi (depending on the cell type) at normal physiological temperatures." Higher pressures
are required to retard significantly the synthesis of DNA, RNA, or proteins.",
Therriiodynarnic Studies
The InouC simple equilibrium model can be formulated from Equation 2
i,
The equilibrium
for the spindle at metaphase, in the following
constant is a function of temperature and pressure:
where AGIO = A H - TAS, AGIO being the difference in standard Gibbs free
Salmon : Assembly Thermodynamics of Spindle Microtubules
387
energy at atmospheric pressure, A H the difference in enthalpy, AS the difference
in entropy, and AV the difference in molar volume for the microtubule subunits
between the polymerized and unpolymerized state. T is measured in degrees
Kelvin, P in psi. At constant temperature, AGIO is a constant and for constant
AV a plot of In K versus pressure will be a linear function given by:
In K = - ( P- 14.7) A V / R T - AG,O/RT
(4)
By rearranging Equation 3, the spindle BR is given by:
The simple equilibrium model predicts the pressure dependence of the
equilibrium spindle BR data surprisingly well over the temperature range
4 and 5). Analysis at the three constant temperatures yields
studied (FIGURES
a large positive value of AV, as expected. The value of AV is approximately
400 ml/mol per polymerizing microtubule subunit (as determined from Equa5 ) . The small differences in AV
tion 4 and the thermodynamic plot in FIGURE
between the three temperatures can be accounted for on experimental grounds,
rather than requiring a temperature dependence of
Alternative equilibrium models derived from considering the order of the
forward and reverse kinetic reactions have also been treated in a manner
4-
I
-
1
P-.
1
2
1
1
4
1
1
6
1
1
1
1
8 R + 2
TIME (min)
1
1
4
1
1
6
1
1
8
1
1
f
3
1
10
FIGURE
2. Comparison of kinetic changes in half-spindle BR and spindle interpolar
length during and after a 2,000 psi pressurization of a metaphase-arrested spindle of
Chaefoprerrcs pergamcrifaceous. Notice that changes in BR occur much faster than
changes in length. The curves were fitted by eye, and the time (in min) is set at zero
for the time of pressurization and pressure release.
Annals New
388
York
Academy of Sciences
parallel to the above analysis, while retaining InouC's assumptions that therc
are a fixed spindle tubulin pool size at metaphase, a proportionality between
measured BR and the amount of polymerized tubulin, a single equilibrium
A
-3.0-
0,
v)
m
-
m
a
0,
n
P
m
0
m
w
n
z
\
hl.0v)
\
\
\
I
10
20
I
30
I
40
I
\
\
1
50
60
8
-3.0
E
-
C
a
m20-
w
6z
h1.0-
'
v , /
I
f
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FIGURE3. Changes in spindle BR during the course of mitosis and changes that
result from the application of pressure in the first mitotic spindle of S. droebochiertsis
a 8" C. (- - - -)=Changes in average half-spindle BR (modified from Stephens ")
following nuclear membrane breakdown through mitosis at atmospheric pressure.
(A) 2,500 psi was applied at the beginning of metaphase and held constant through
the rest of mitosis. The small, weakly birefringent spindle (FIGURE1 ) still progressed through mitosis, with the characteristic changes in the spindle BR acompanying the changes in stage. (B) Changes in spindle BR during the application and release of pressure during metaphase ((3-0) and during anaphase (A-A).
Pressure appeared to have little effect on the stage-timing unless held for a prolonged
period, as in A.
constant and constant magnitudes of AH, AS, and AF over the pressure and
temperature range studied.46 A model based on first-order polymerization and
second-order depolymerization (K= [B]/[A, - BIZ),similar to the linear condensation treatment of in vitro TMV protein assembly by Lauffer et al.," fits
lo00
PRESSURE - psi
1500
2000
-
2500
3000
FIGURE
4. Equilibrium spindle BR as a function of pressure a t constant temperatures of 22" C, 17.5" C, and 14.5" C for the metaphase-arrested spindle of Chaefopfenu. Data include measurements from spindles in whole eggs and from spindles in small yolk-free
egg fragments produced by centrifugation methods. In a single experiment, pressure was increased and held constant until the spindle reached equilibrium and the measurement made. Then the pressure was increased further in similar steps until the spindle was
almost completely depolymerized. The pressure was then returned to atmospheric, and the spindle allowed t o recover. The process was
often repeated for different magnitudes of pressure increment on the same spindle. The solid lines are theoretical curves derived from
5 ) 'O and Equations 3 and 5 in the text.
the simple equilibrium model, using the derived thermodynamic parameters (FIGURE
0
220
22O Pressure Steps ,1000 psi
x 220Fragmemt Spindle
A 1Z5O
0 14.5O
Theory AV=400ml/mole
0
\o
OQ
v,
1
5.
z
5
m
z.
a
E
z
Y
3
CD
v1
P
Annals New York Academy of Sciences
390
A
t
t
003'
1
I
1
1
2
1
PSI x 10.3
1
1
3
FIGURE
5. Equilibrium constant based on the simple equilibrium model plotted as
a function of pressure for the metaphase-arrested spindle of Chaetopterus. The equili4, according to the relation
brium constant was found for each data point in FIGURE
K_=[B]/[Ao-B] for the previously determined value of Ao=5.8.'' The values of
AV were determined from the slopes and standard errors of the least-square lines
through the data points for each experiment temperaure, using Equation 4 in the
text.'O
Salmon : Assembly Thermodynamics of Spindle Microtubules
39 1
the Chaetopterus pressure and temperature data a b o u t as well as the simple
equilibrium model..*6 This analysis yields a value for A V of about 525 ml/mol
for the three temperatures. A model based on nucleated condensation or end
polymerization (K = 1 / [ A , - B J ) , similar to Oosawa’s modelfor actin polymerization in vifro,“’. I!’ also yields a large positive value of AV (100-200 ml/
mol). In this model the equilibrium constant depends only on the reciprocal of
the unpolymerized subunit concentration, but the model does not fit the data
well at 22°C when assuming only a single equilibrium constant governs the
polymerization.
Although the Chaetopterus pressure and temperature data are fitted about
equally by either the simple equilibrium or linear condensation models,
StephensIs has found that only the simple equilibrium model fits optimally
the temperature dependence of spindle BR for two different pool sizes in the
active metaphase spindles of S. droebachiensis. Since S. droebachiensis is
adapted for reproduction and development at unusually cold temperatures
(between 0” and 10”C ) , it was initially thought that high magnitudes of
pressure might be required to produce complete spindle depolymerization.
Near the normal developmental temperature (8” C), however, only about
4,000 psi is needed. Further thermodynasic analysis based on the simple
equilibrium model yielded a value for AV of 343 ml/mol for the spindle
assembly.
The large value of A F and the low magnitude of AGIO for the monomerpolymer equilibrium at normal physiological temperatures readily accounts
for the high sensitivity of spindle microtubules to depolymerization by pressure.
The large values of positive entropy and of the molar volume change during
polymerization appear to result from many moles of structured or constricted
water being released as a number of hydrophobic or ionic residues are transferred into intermolecular bonds during polymerization.’?, l J .
It is highly
probable that the interaction between tubulin subunits and solvent contributes
substantially to the stability of spindle microtubules. Modification of the cell
medium with either heavy water or low dielectric solvents such as hexylene
glycol does promote spindle polymerization.s*
Although the normal thermodynamic characteristics of the spindle assembly
process in vivo and the major subunit bonding characteristics have now been
established, the actual molecular mechanisms of polymerization and the identity
of the cellular factors that control the normal assembly and disassembly of the
spindle during mitosis remain largely a mystery. During the course of mitosis
the half-spindle BR changes in a characteristic
lG. lli
Following
breakdown of the nuclear membrane, there is an initial rapid rise in BR as
the central spindle forms, then a slower increase through prometaphase to an
approximately constant plateau at metaphase (FIGURE
3 ) . At about the time
that anaphase begins the BR rises rapidly, then it decays through the course
of anaphase. In terms of Inoui’s model, changes in the microtubule assembly
throughout mitosis should be reflected in the changes in spindle BR, as given by:
A time dependency (t) for A,, is included because spindle assembly and disassembly may be controlled by “activation” or “inactivation” of part of the
total cellular tubulin pool into subunits capable of being assembled into microtubules under otherwise constant conditions. The equilibrium constant, K’( t ) ,
392
Annals New York Academy of Sciences
may also be a function of physiological factors that change as mitosis proceeds
under constant temperature and pressure.
To support the former concept, Stephens x has shown that there is
apparently a process of tubulin activation, sensitive to temperature during
prophase but not metaphase, that governs the size of the spindle tubulin pool
at metaphase. Deactivation of the pool is thought to contribute to spindle fiber
depolymerization and chromosome-to-pole movement during anaphase.s,
Weisenberg3??5 2 has found that the polymeric form of tubulin varies between
a granular particle during interphase, a soluble form at about the time of
nuclear membrane breakdown, and the microtubules of the spindle at metaphase.
He has suggested that this particle may be a storage form of the spindle tubulin.
Pressurization of the spindle in early anaphase produces a loss of spindle
BR which, when pressure is released, recovers to a magnitude characteristic of
the decaying BR of a control spindle at that time under atmospheric pressure
(FIGURE
3 B ) . The monomer-polymer equilibrium still exists in anaphase, but
sufficient evidence is not yet available to distinguish convincingly between
contributions to the control of polymerization either by changes in the spindle
tubulin pool or changes in physiological factors that affect the apparent equilibrium constant.
The optimum fit of the simple equilibrium model to the BR data does not
necessarily imply the physically unlikely situation of a first-order polymerizationdepolymerization mechanism. It is indicative, however, of the probable differences in the physiological conditions and mechanisms that govern assembly of
the microtubules in the spindle from those involved with assembly under testtube conditions, which appears to be characterized by nucleated condensation.eEve9 It is not known whether the polymerizing subunit is the tubulin dimer
or higher polymeric
0 3 , 5R,
The helical polymerization of TMV
protein in vitro does involve several intermediate aggregates,",
53 whose
participation in the rod assembly appears to depend on slight changes in the
ionic strength and pH. Whether microtubules can assemble only at their ends
hl,
Iiy The kinetic
or possibly all along their lengths is also not at all
data discussed below and the data from others ,I1indicate that tubules can have
different stabilities, depending on their location within the spindle; this implies
that more than a single equilibrium constant may actually govern the assembly
reaction. These stability differences may be due to different types of tubulin,
but local distributions or gradients of ions such as calcium 3 4 , may be responsible for this differential stability, as well as producing net fluxes of tubulin
along the microtubules of the spindle fibers.', R , 6 G
Nucleation is an important feature of microtubule polymerization in the
spindle (as judged by the normal spatial distribution and orientation of microtubules within the spindle,', fi and by the delay in spindle recovery following
release of high magnitudes of pressure"'). It is becoming increasingly important that we should better understand the molecular identity of nucleation
or "orientation" centers at the kinetochores and the poles, and their activity i n
controlling the apparent spindle pool size locally, in anchoring the microtubules,
and in regulating local and selective polymerization and depolymerization of
the microtubules within the spindle, particularly with respect to mechanisms of
chromosome movement.7. E, 46, 507 70
"Ii
s3s
39
393
Salmon: Assembly Thermodynamics of Spindle Microtubules
Kinetic Studies
After pressurization of Chuetoplerirs oocytes, spindle length approaches
2 ) . BR decay also
equilibrium as a roughly exponential function (FIGURE
appears to be approximately exponential. A more detailed analysis, however,
6) : an initial, rapid
has shown that the decay consists of two phases (FIGURE
phase of BR decay and a second phase of much slower BR decay during which
the majority of spindle shortening takes place I t (see also FIGURE
11). Both
o
2500 psi
A
3000 psi
4000 psi
m5500 psi
0 7200 psi
8
1
2
3
MINUTES AFTER PRESSWllZATlON
4
FIGURE
6. Changes in half-spindle BR as a function of pressure in the metaphasearrested spindle of Clruetopferrts. The BR measurements were normalized for each
experiment by division with the atmospheric value of the spindle BR measured before
pressurization (3.65-CO.2 nm, S.D.). The data were obtained from densitometric
analysis of 16-mmtime-lapse film records of repetitive experiments on a single spindle. The data for 5,500 psi and 7,200 psi are not quantitatively accurate, due to the
interference of stress birefringence in the chamber windows at these pressures."
the rates of spindle shortening and the decay of BR in the initial, linear phase,
increase progressively with pressure. For pressures of 6,000 psi or greater, only
the BR decays; there is no spindle shortening. When the normalized initial rate
of BR decay is plotted against the normalized initial shortening rate f o r pressures of 4,000 psi or less, they fall o n a straight line (FIGURE
7). Regardless
of the pressure, the initial rate of BR decay is about 5.2 times faster than the
initial rate of spindle shortening. Except f o r a possible delay in reappearance,
spindle recovery from all pressures below 6,000 psi is similar to the pattern
2. The BR rises rapidly, then remains nearly constant as
shown in FIGURE
Annals New York Academy of Sciences
394
the spindle slowly elongates and returns to its normal metaphase interpolar
length.
I have suggested the following hypothesis to explain the kinetic data. The
length of the longest microtubules controls pole-to-pole and half-spindle length,
and the stability of a microtubule depends on the number of its “free ends.”
(“Free ends” are ends not attached or engaged at the kinetochores or pole
regions.) According to this hypothesis, a microtubule with one free end would
depolymerize or polymerize more rapidly than a microtubule with two attached
I
/
FIGURE
7. Correlation of the normalized initial BR decay rate to the normalized
initial shortening rate (-)
and normalized velocity of induced chromosome movement (- - - -) under a given pressure of 4,000 psi or less on Cliaetopterzcs spindles.
The data were normalized by dividing respectively the rates of initial spindle BR decay and shortening by their previously measured atmospheric equilibrium values, and
by dividing the velocities of induced chromosome movement by the initial distances
of the chromosomes from the cell surface. The lines were fitted by the method of
least squares. The intercepts are 0.0, since no changes in spindle BR or interpolar
length occur without pressurization.
ends. Microtubules may be capable of exchanging subunits all along their
4 G , 51* 5 7 , 69 but the loss or addition of subunits within a microtubule is assumed to proceed at a slower rate than polymerization or depolymerization at a free end.
In support of this hypothesis, serial sections of various spindles examined
with electron microscopy have indicated that only 10 to 40% of the continuous
fiber microtubules extend from pole to pole. Most extend from one pole toward
the other, ending and overlapping in the metaphase plate region.fi9-G2Thus
Salmon : Assembly Thermodynamics of Spindle Microtubules
395
they have at least one “free end.” In chromosomal fibers, a higher percentage
of microtubules appears to extend all the way from the kinetochores to the poles.
When pressure is applied, the large number of continuous fiber microtubules
that have free ends in either of the half-spindle regions will depolymerize
6 ) . The spindle
rapidly, producing the majority of the BR decay (FIGURE
shortening occurs at nearly constant BR, which indicates that there is a nearly
constant number of spindle fiber microtubules. The slower rate of spindle
shortening at constant microtubule number indicates the slower rate of depolymerization for microtubules engaged at both ends. Return to atmospheric
pressure reverses the process. Initially, most microtubules have a free end.
The fibers repolymerize and grow rapidly until they enter the polar region. Thus
the BR rises rapidly before much elongation occurs (FIGURE2 ) . The spindle
then slowly elongates, with nearly constant microtubule number, as the interpolar and kinetochore-to-pole microtubules slowly polymerize and grow longer.
The hypothesis proposed here does not require the assumption that different
types of microtubules (or different types of microtubule subunits) are present
to explain differences in the stability of spindle fibers; stability is determined
only by the microtubule’s location and the engagement of its ends. The constant
proportionality of the normalized initial BR decay rate and normalized initial
shortening rate over the pressure range examined indicates that all the microtubules have the same molar volume change of activation for subunit dissociation. The difference in the rates must be the result of differences in other factors
that affect subunit bonding. That BR decays but the spindle does not shorten
in response to pressures of 6,000 psi or higher indicates that high pressures,
like high doses of colchicine .I or very low temperatures,T cause the microtubules
to depolymerize so rapidly that they lose their mechanical integrity.
ROLEOF MICROTUBULE
DEPOLYMERIZATION
I N CHROMOSOME
MOVEMENT
The function of the microtubules and the means of force generation in
chromosome movement are not yet clear. The various molecular theories of
mitotic movements fall into two groups: those that involve controlled shortening
and lengthening of spindle fiber microtubules to produce forces for chromosome
movement,G-8sO 7 and those that involve the fiber assembly indirectly.
The latter theories propose that shearing or sliding mechanisms that act on
the fibers generate the forces for chromosome movement. Ostergren (i3 proposed that the chromosomal fibers initiate shearing forces in the direction of
the poles, and these forces act upon the fibers to produce poleward movement.
I n providing molecular mechanisms for the above ideas, Subirana suggested
that the spindle fiber microtubules have a characteristic polarity, and that a
myosin-actin-like interaction between the microtubules and spindle matrix
produces shearing forces, or else that microtubules have myosin-like arms that
give them the capability of “swimming.” McIntosh liGextended and modified
this concept by proposing that forces for moving chromosomes are produced
by the mechanical action of cross-bridges between microtubules. Bajer Oi has
hypothesized that lateral interaction (“zipping”) between nonparallel microtubules is the motive force for chromosome movement, and that sliding forces
produced by cross-bridging molecules are not required. Experimental evidence
to support the above theories has been based primarily on interpretations of
questionable microtubule distributions, determined from electron micrographs
396
Annals N e w Y o r k Academy of Sciences
of spindles in selected organisms. Thus far no one has convincingly identified
dynein, myosin, or actin in the spindle.
InouC c-s and Dietz 6i maintain that chromosome movement is inherently
or causally related to changes in the assembly of microtubules of the spindle
fibers: the chromosomes are initially positioned to the metaphase plate, and
later are separated by localized and selective depolymerization and polymerization of the chromosomal and continuous fiber microtubules.
The principal experimental evidence for the above hypothesis has been the
demonstration that following slow depolymerization of metaphase-arrested
2 1 the chromospindles with colchicine,' low temperature,', or other
somes moved from their metaphase plate position in the cytoplasm to the cell
surface near the position of the strongly anchored spindle pole. After the
removal of colchicine o r warming, the chromosomes returned to their original
metaphase position. Conversely, increased spindle polymerization produced by
D 2 0 was manifested by increased spindle BR, as well as increased interpolar
distance.
a
b
FIGURE
8. Induced movement of the chromosomes (9 bivalents) in the metaphasearrested spindle of Chaetopterus as the spindle depolymerizes then repolymerizes following the application (a) and release ( b ) of 3,000 psi. Time in minutes is indicated
on each frame, with the time of pressurization and the time of pressure release set at
zero. Generally noticeable as the chromosomes moved toward the surface was a
transient decrease in curvature or dimpling of the cell surface where the spindle
,aster was anchored. After pressure release, the surface bulged out at the point of
aster attachment, the bulge disappearing as the spindle recovered. Bar= 10 pm.
I
-
0
20
0
1
1
1
1
1
I
l
l
.
1
1
1
1
1
I
I
* , o Chromosome to Surface
Half - Spindle Length
Correlntion of Induced Chroniosorne Movernent to
Changes in Microtubule Assembly
I have recently examined the relationship between movement of chromosomes and transient changes in microtubule assembly induced by pressure in
the meiotic metaphase-arrested spindle of Chaetopterus." 54 Depolymerization
and repolymerization of the spindle produced chromosome movement, as pre8 ) . Movement occurred for moderate rates of depolymerization
dicted (FIGURE
induced by pressurizations of less than 6,000 psi at 22" C . Above 6,000 psi,
depolymerization was rapid, the spindle did not shorten, and the chromosomes
did not move toward the cell surface. Instead, the position of the chromosomes
became random, as if they were freed from their attachments. These general
events are identical to those that occurred when cold or colchicine were used
as the depolymerizing agents. 1,
Kinetic changes in the birefringent spindle structure were correlated with
the movement of chromosomes for moderate rates of depolymerization (FIGURE
9 ) . Under pressures of 4,000 psi or less, the chromosomes tended to move as
a group toward the pole anchored at the cell surface (FIGURE
8 ) . They main-
398
Annals New York Academy of Sciences
tained an approximately equatorial position as the spindle shortened. The
spindle, with its chromosomes, also moved clqser to the cell surface as the
attached aster shortened.1t,5 5 After atmospheric pressure was restored (FIGURE
9 ) , the above process was essentially reversed.
A closer analysis of individual chromosome movement revealed that although the chromosomes appeared to move as a group during lower pressurizations, they actually moved independently.5' This was particularly evident in
experiments with higher pressures (between 5,000 and 6,000 psi), in which not
all chromosomes moved all the way to the cell surface. The apparently independent movement of individual chromosomes indicates that the forces producing movement are transmitted to the chromosomes individually.
The chromosomes, when they moved, moved at progressively higher velocities with increasing magnitudes of pressure to 6,000 psi. The rate of movement,
taken from the approximately constant velocity regions of the curves illustrated
in FIGURE
10, increased from approximately 4 pm/min at 2,500 psi to about
10 pmlmin at 4,000 psi, then to 17 pm/min at 5,500 psi. This latter value is
two to three times the normal anaphase velocity at 22" C . Increases in the rate
of chromosome movement are also linearly correlated with increases in both
the rate of spindle shortening and the rate of spindle BR decay (FIGURE
7),
the normalized rate of chromosome movement being 1.1 times faster than the
normalized rate of half-spindle shortening and 5.1 times slower than the normalized half-spindle BR decay rate.
This correlation strongly supports the hypothesis stated previously, that the
lengths of the longest microtubules control the pole-to-pole and kinetochore-topole distances. Whether or not the depolymerization of the microtubules alone
is responsible for the forces that produce chromosome movement, the rate of
depolymerization and shortening, or the rate of polymerization and elongation,
appears t o regulate the velocity of the chromosomes (provided that the rate
of depolymerization is not excessive). The loss of induced chromosome movement and the spindle shortening under high rates of depolymerization indicate
that the microtubules are required for proper force transmission.
Overstabilization of mitotic spindles with either high concentrations of
D2O,I38i o low concentrations of hexylene glycol,5o or high temperature,".
blocks anaphase. Fuseler l Ghas recently demonstrated that the rate of spindle
BR decay during anaphase increases progressively with higher temperatures, as
does the chromosome velocity. Over a wide temperature range the initial rate
of anaphase chromosome movement was shown to be proportional to the initial
rate of spindle BR decay, and independent of the absolute magnitude of BR.
Whether this linear correlation is causal or whether both variables are controlled
by the same factor (or factors) that changes as the mitotic stage advances
remains to be determined.
Independence of Chroriiosorne Velocity cind Microtiihule
Number or Chromosome Drag Force
Although the rate of change of microtubule assembly correlates highly
with chromosome velocity, there is, in general, little correlation between
chromosome velocity and either microtubule number or drag force. For example, when metaphase spindles were depolymerized with pressure, the high
rates of chromosome movement occurred when little BR was detectable, that
Salmon : Assembly Thermodynamics of Spindle Microtubules
399
is, there were few microtubules (FIGURE1 1 ) , Although electron microscopy
studies will be required to determine the exact number and location of microtubules under these conditions, abnormally rapid velocities have been observed
I
O
2500 psi
i
3
2
1
0
O
-
5000psi
5500pri
0
L
0
I
I
1
I
TIME- min
I
2
I
I
3
FIGURE
10. Kinetic changes in the position of the chromosomes with respect to the
cell surface as a function of increasing magnitudes of pressure for Chaetopferrrs
spindles. Time of pressurization is set at zero. Except for 5,500 psi the chromosome
position data represent the average position of a group of 3 or more chromosomes
(FIGURE
8). The chromosome position data for 5,500 psi represent one chromosome,
since not all the chromosomes moved all the way to the cell surface. In 6 experirncnts with 6,000 psi or higher pressure, no net chromosome movement occurred.
As the pressure increased, there was a tendency for the onset of chromosome movement to he delayed. This appears to be becatise the attached aster initially pulls
away from the cell surface as the microtubules rapidly depolymerize and the spindle
shortens. Then the aster reattaches and moves hack closer to the cell surface, pulling
the shortening spindle and chromosomes with it.
in other systems under conditions that involved low numbers of microtubules.l*
Since the force and energy requirements for chromosome movement appear
very low,'. 2 7 ~7 2 it is reasonable to assume that there are adequate forces to
produce rapid movements of the chromosomes even with very low numbers
400
Annals New
York Academy of Sciences
of microtubules. Why then are there so many more microtubules in the spindle
than appear to be necessary f o r moving chromosomes? One possible answer
may be, as suggested earlier, that they are required to adequately control the
rate of chromosome movement. For a given amount of microtubule depolymerization, overall fiber shortening and chromosome movement would be slower
and more easily controlled if there were many microtubules than if there were
J
TIME - min.
FIGURE11. Typical kinetic changes in the position of the chromosomes with respect to the cell surface, in spindle BR, and in half the spindle pole-to-pole distance
for Chaetopterirs spindles following application of 4,000 psi. Note that rapid induced
chromosome movement and spindle shortening take place at low magnitudes of spindle BR. There are less than 250 microtubules in the half-spindle at this time, compared with 5,000 at atmospheric pressure (as determined from the measured BR and
theoretical form birefringence calculations ’, ”) .
very few. This rate-limiting feature would explain the normal “velocity loadindependence” of anaphase chromosome movement. Anaphase chromosome
velocities d o not depend o n chromosome size, viscosity, or chromosome stretching in unseparated bivalents over wide ranges.2:. :*, i4 As long as the forces for
moving the chromosomes are sufficiently strong, the rates at which the chromosomes move will depend only o n the rate at which the spindle fiber microtubules
change their length.
Salmon : Assembly Thermodynamics of Spindle Microtubules
401
Microtubule Anchorage
The movement of chromosomes in the Chrretopterus oocytes, as the spindle
depolymerizes or polymerizes, appears to be a direct consequence of the firm
attachment of one of the spindle asters to the cell surface and the loose attachment of the other aster in the cytoplasm or o n the opposite cell surface. In
other cells in which the spindle is situated more nearly in the center of the cell,
depolymerization by either low temperature,I1’ colchicine,75 or high pressure
(note FIGURE
1 ) causes the poles to collapse toward the metaphase plate, and
there is no net chromosome movement. Although polarized light studies ?B
and electron micrographs 7 i have shown that the aster and continuous fiber
microtubules are depolymerized more easily than are the chromosomal fibers,
the asters of the Chnetopterus spindle, although weak, remained visible during
spindle shortening for pressures u p to 4,000 psi.
The anchorage of the spindle to the surface is not static, but reflects a
dynamic polymerization-depolymerization of the aster microtubules.” During
depolymerization under pressure, the attached aster appeared to shorten as
the spindle moved closer to the cell surface. After pressure was released, the
aster enlarged as the spindle moved back into the cytoplasm. At atmospheric
pressure the spindle was also occasionally observed to move rapidly toward
the membrane, then just as suddenly back into the cytoplasm. More frequently
seen, however, was a rapid and erratic pivoting of the spindle about the
attached pole and an exchange of the pole attached to the surface.
Little is known about how astral microtubules or spindle fiber microtubules
are actually anchored, but micromanipulation studies of meiosis I grasshopper
spermatocytes by Nicklas and Staehly 7’ demonstrated that the chromosomes
are individually anchored at the kinetochores and at the spindle poles. Their
observations suggested that the chromosomal fiber could be characterized as
a “string or thin wire” attached only at the kinetochore and the pole. Although
microtubules appear to attach directly to the kinetochores,’. ,’, the microtubules that begin and end in the centrosphere d o not attach to the centrioles
located there, but appear simply to end in a region near the centrioles.7!’ Specific
sites responsible for polymerizing, depolymerizing, or anchoring the microtubules in the centrosphere still have not been convincingly identified. Some
lateral interaction or cross-bridging may occur between the chromosomal fiber
microtubules and the microtubules of the continuous spindle,65* but the
micromanipulation evidence strongly suggests that cross-bridging throughout
the spindle (as originally postulated by McIntosho5) does not play a major
role in anchoring the chromosomes to the spindle. It would be relevant to
know the mechanical characteristics of the anchorage of spindle fibers in anastral
spindles.
Mechunisnis of Force Production
The chromosome movement data discussed here have demonstrated that:
( 1 ) microtubules transmit the forces that produce movement to the chromosomes; ( 2 ) the rate of chromosome movement is linearly correlated with the
rate of change of spindle microtubule assembly and microtubule length; and
( 3 ) chromosome velocity is independent of microtubule number, as well as of
chromosome drag force. The last result, in particular, is difficult to explain in
402
Annals New York Academy of Sciences
terms of Bajer’s “zipper” hypothesis of chromosome movement.G7 His hypothesis assumes that continuous cycles of “lateral-interaction” between nonparallel microtubules of the chromosome and nonkinetochore fibers nlorie can
provide the basic mechanical requirements that govern chromosome movement.
Higher numbers of spindle microtubules should be correlated with higher rates
of chromosome movement, since the frequency of postulated “lateral-interactions” would be increased. Clearly, this is not the case.
The popular cross-bridge theory of force production for chromosome movement, postulated by McIntosh G5, and modified by Nicklas,’? 81 is not required
to explain the data presented here. The data themselves d o not exclude the
model, since changes in microtubule length could provide regulation of the
rates of chromosome movement consistent with the postulates of the model.
It is, in fact, difficult to differentiate experimentally between cross-bridge and
depolymerization-polymerization mechanisms of force production. Proposed
forces generated by the cross-bridges are transmitted by the microtubules. Experimentally induced changes in the assembly of the microtubules could, therefore, artificially produce changes in the force balance. It should be noted,
however, that several essential features of the McIntosh cross-bridge model 6 5 , Gfi
are not typical of many cells. The model indicates that early anaphase chromosome-to-pole and spindle interpolar elongation should occur simultaneously.
This is not true in many cell types; often chromosome-to-pole movement occurs
first, then the spindle elongates.’, i2-i.1, R I - s C , A chromosome artificially dislocated back across the spindle equatorial region in late anaphase should not
be able to return to its original pole, but it can.’. x1 Changes in the distribution
of microtubules during anaphase predicted by the model have not been found
in several cell types.23~
In order to overcome these difficulties and others, Nicklas 1 $ h 1 has proposed
an alternative sliding microtubule model. This model, however, lacks the simplicity and specific predictive capability of the model postulated by McIntosh.fi5
There is no direct evidence, at this moment, that any form of sliding mechanism
produces chromosome movement.x1
On the other hand, the more general depolymerization-polymerizationhypothesis of force production 6 - p - si explains most simply the chromosome movement data presented here, as well as the normal rapid pivoting and translation
of spindles and the transient deformations of the cell surface during depolymerization and repolymerization of Chczetopterus spindles. Polymerization of
microtubules does produce pushing forces, as is shown by the movement of
chromosomes that accompanies regrowth of the spindle after pressure is released (note FIGURES
2 and 8 ) . If they are not excessive, increasing rates of
microtubule depolymerization (induced by pressure, colchicine, or low temperature) do produce increasing rates of chromosome movement. It is significant
that the rates of chromosome movement induced by lowered temperatures are
nearly identical to those induced by colchicine or pressure.’. i. 5 q , h i The dosages
of colchicine p? and magnitudes of pressure ’:<* h R employed hardly affect the
dynein ATPase activity and motility of cilia and flagella, whereas the low
temperatures used markedly reduce both.“’ Establishment of a mechanism of
force production by controlled microtubule polymerization-depolymerization,
in particular for chromosome-to-pole movement, depends on demonstrating
that spindle fiber microtubules can add and subtract subunits without losing
their mechanical integrity. Evidence presented at this conference indicates that
li9
microtubules may be capable of depolymerizing all along their
Salmon : Assembly Thermodynamics of Spindle Microtubules
403
The establishment of a depolymerization-polymerizationmechanism will also
depend critically on identifying the “orienting” centers at the kinetochores and
the poles,G-Rand how they act to anchor the microtubules and to control polymerization and depolymerization of microtubules both locally and selectively. At
this time, controlled polymerization and depolymerization of the microtubules
does appear to play an important role in transmitting and regulating the balance
of forces in the spindle and in controlling the rates at which chromosomes move.
SUMMARY
In this paper I have presented results of experiments in which spindle
microtubules were depolymerized by hydrostatic pressure, in order to examine
the InouC dynamic equilibrium concept of spindle assembly and the possible
role of microtubule depolymerization-polymerization in the movement of chromosomes. Using a newly developed optical hydrostatic pressure chamber, I
investigated with polarization microscopy the quantitative effects of pressure
on the polymerization of spindle microtubules and, with phase contrast microscopy, the relationship of pressure-induced spindle microtubule depolymerization
to chromosome movement in living cells. From results of earlier experiments,
principally those of JnouC et al. with low temperature and colchicine as microtubule-depolymerizing agents, and from results of my own research, I have
concluded that: ( 1 ) spindle fiber microtubules are sensitive to depolymerization
by pressure (3000-7000 psi), spindle microtubules do exist in a labile equilibrium with a pool of subunits, and the InouC simple equilibrium model
does predict changes in spindle microtubule assembly at metaphase induced by
pressure; ( 2 ) the stability of microtubules depends on the number of “attached
ends;” (3) the longest interpolar microtubules and the longest chromosomal
fiber microtubules regulate the spindle interpolar length and the chromosometo-pole positions; (4) chromosome velocity is independent of the number of
spindle microtubules, as well as of the drag force of the chromosomes; (5)
the chromosomal fiber microtubules transmit the forces between the poles and
between the chromosomes and the poles; and (6) polymerization of microtubules does produce pushing forces and, if controlled microtubule depolymerization does not actually produce pulling forces, at least it governs the
velocity of chromosome-to-pole movement.
ACKNOWLEDGMENTS
I wish to thank Drs. S. InouC and R. E. Stephens for their valuable discussions and criticisms, and for providing laboratory facilities and equipment.
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