AMER. ZOOL.. 15:649-660 (1975).
Some Factors Involved in the Control of Microtubule Assembly in Sea Urchins
JOSEPH BRYAN
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19174
SYNOPSIS. Some recent evidence is discussed which suggests that the control of microtubule
assembly during the formation of the mitotic apparatus in sea urchins involves three factors.
These include: nucleation sites such as the kinetochores, a mechanism for regulating
intracellular calcium, and a heat stable substance which prevents spontaneous, nonnucleated microtubule polymerization.
The purpose of this paper is to provide a
rather brief review of the control of microtubule assembly, and in the context of this
symposium, indicate how echinoderms
have been useful in studying this problem.
The area of microtubule biology has been
so thoroughly reviewed in the last few
years, including a major symposium to be
published in early 1975 by the New York
Academy of Science, that it seems unwarranted to recover old ground. I would like,
therefore, to review some of the recent,
more speculative work with echinoderms
and other organisms on the factors involved in the regulation of microtubule assembly during mitosis. Some of the problems involved in the control of microtubule
assembly are apparent if we consider the
formation of the mitotic apparatus (MA) in
echinoderms. The MA is a transient cellular organelle whose major purpose is to
equally partition the chromosomes after
DNA replication. Microtubules are the
major fibrous component of the MA. Bundles of microtubules are known to comprise
the astral, chromosomal, and continuous
spindle "fibers" seen in mitotic cells under
phase contrast microscopy or in fixed and
stain mitotic cells. In the polarization microscope a similar pattern of birefringent
Various aspects of the work reported here were
supported by National Science Foundation Grant
GB-3287X. Some of the work was done at the Friday
Harbor Laboratories of the University of Washington.
I am grateful to Dr. Dennis Willows for the opportunity to work there and to Dr. Arthur Whiteley for material and equipment. The technical assistance of Mr.
Philip Barr and Mr. William Cook was greatly appreciated.
fibers is seen in the MA. The birefringence
is predominately form birefringence, due
to oriented microtubules (Sato, 1975). The
echinoderm MA is particularly suited to
studies on microtubule formation since
there appears to be a relatively large pool of
tubulin in the echinoderm egg, eliminating
the necessity for total de novo synthesis. (See
Mazia, 1961, and Inoue and Sato, 1967, for
the arguments for preformed pools. See
Burnside et al., 1973, for a chemical estimate of the tubulin pool size in Arbacia.
Finally see Raff, 1975, for further information on tubulin pools.)
From the morphological descriptions of
spindle fibers (see Mazia, 1961; Harris,
1961, 1962) and particularly the studies on
the birefringence changes of these fibers
during mitosis (Inoue and Sato, 1967), two
things regarding microtubule formation
seem clear. First, beginning, in the sea urchin egg, with the separation of centrioles
or centers in prophase, there is an orderly
temporal sequence for the polymerization of
tubulin into microtubules and a subsequent
depolymerization beginning in late anaphase and extending into telophase. During this period, it is generally accepted that
an equilibrium exists between free tubulin
in the cellular pool and structured tubulin
in microtubules. It is not yet clear if an
individual microtubule forms and then remains intact during this entire time or if an
individual tubule is in a state of flux, with
tubulin subunits constantly "cycling"
through it. Rebhun and his co-workers
(1975) have proposed that tubulin is constantly cycling through the microtubules in
a mitotic apparatus, but it is not yet clear
649
650
JOSEPH BRYAN
how this can occur at the molecular level.
The idea is, however, potentially important
in that it would be useful in discriminating
between sliding models (Mclntosh et al.,
1969), assembly-disassembly models (Inoue and Sato, 1967), and "Zipper-models"
(Bajer, 1973) of chromosome movement.
A second feature of the spindle fibers or
bundles of microtubules is their apparent
spatial order. The polymerization of microtubules during the formation of the MA is
not a random process, but rather appears to
be highly localized with microtubule
growth being initiated at quite specific
places: on the chromosomes at the
kinetochore and at the poles in the region
around the centrioles. The kinetochores
define the orientation and location of the
chromosomal fibers, while the centers
define the continuous fibers and astral
fibers (see Inoue and Sato, 1967; Mazia,
1961). In general there do not appear to be
significant numbers of randomly oriented
microtubules.
The positioning of the kinetochores and
centers is insufficient to account for the
overall three-dimensional architecture of
the spindle, for example, the curvature of
microtubules. Mclntosh et al. (1969) have
proposed that at least one additional factor,
the proteins bridging MT's, are also involved in determining the overall order of
the microtubules in the MA. The extensive
endoplasmic reticulum observed by Harris
(1961, 1962), particularly at the poles, may
also play a role here.
The recent work on in vitro assembly of
microtubules from mammalian brain, and
the attempts to obtain assembly in other
cells, suggest that the assembly-disassembly
and positioning of microtubules during
mitosis appear to be the result of the interplay of three factors. These include: (i)
The sites which nucleate or initiate the assembly of microtubules. In the echinoderm
MA these are the kinetochores on the
chromosomes and the centrioles or
pericentriolar material, (ii) A system for
regulating the assembly and disassembly of
tubulin once microtubule formation has
been initiated. There is some evidence that
this system may be involved in the regulation of cytoplasmic Ca2+ concentrations.
(iii) A factor for suppressing the spontaneous assembly of microtubules directly from
tubulin.
Since the evidence for each of these three
factors depends in part on recent work on
microtubule structure and on tubulin assembly in vitro, a brief review seems relevant.
A BRIEF SUMMARY OF MICROTUBULE STRUCTURE
Microtubules are long tubular polymers
composed of relatively small globular protein subunits called tubulins. Two major
classes of tubulin have been defined termed
a- and j8-tubulins (see Bryan, 1974, or Olmsted and Borisy, 1973, for a review). The
biologically important unit is a dimeric
molecule composed of one a and one /3
tubulin (Bryan and Wilson, 1971; Luduena
et al., 1974). In the electron microscope
microtubules appear as long cyclindrical
structures with an outside diameter of 22 to
25 nm and an apparently hollow core 15
nm in diameter. The 4- to 5-nm thick walls
which comprise the tubule have been demonstrated in the Arbacia mitotic apparatus
to be composed of 13 protofilaments each
of which is made from a- and /3-tubulins
(Tilney et al., 1973). While very high resolution electron microscopy has been done
on the flagellar outer doublet microtubules
from Strongylocentrotus purpuratus (Warner
and Satir, 1973; Warner and Meza, 1974),
our most detailed picture of microtubules
comes from work on Trichonympha. Amos
and Klug (1974) have combined high resolution EM, optical diffraction, and computer imaging with the relevant chemical data
to produce a detailed model of a flagellar
microtubule (see Figs. 1, 2, for details). The
model suggests that we can look at a microtubule either as 13 laterally associated
protofilaments or as a helically wound tubular polymer. The analysis of microtubules
from other sources has indicated similar
patterns (Erickson, 1974; 1975).
IN VITRO ASSEMBLY OF BRAIN TUBULIN
Preparations of the cytosol fraction of
brain, a tissue rich in tubulin, will form
CONTROLS OF MICROTUBULE ASSEMBLY
831
2+
outside
8.0nm
0
3.0 6.0 9.0 12.0nm
inside view
centrations, Ca can promote assembly,
while at higher Mg2+ concentrations (5mM),
Ca2+ at 10 /AM, can inhibit assembly (Rosenfeld and Weisenberg, 1974).
"Cycling" procedures for purifying
assembly-competent tubulin from either
porcine or bovine brain have been reported
by Borisy et al. (1974) and Shelanski et al.
(1973). The end product in either procedure contains four proteins (see Fig. 3), the
a- and /8-tubulins and two or more microtubule associated proteins (MAP's) which
may have ATPase activity (Burns and Pollard, 1974; Gaskin et al., 1974) and are responsible for the "decorations" seen on
negatively stained microtubules from in
vitro assembled brain tubulin (Murphy and
Borisy, 1974; Dentler et al., 1975). The as-
outside view
FIG. 1. Arrangement of subunits in a Trichonympha
flagellar microtubule. The top view is a radial section
through the center of an individual protofilament in
the wall of the microtubule. The radial zig-zag arrangement produced by alternating morphological
units is evident. The bottom views are superimposed
"cylindrical sections" through two adjacent protofilaments viewed either from "inside" or "outside" the
microtubule. In each view, a contour line was drawn
outlining the protofilament in the cylindrical section
for a given radius. The inside view was taken from
sections with radii between 7.2 and 9.6 nm, the outside
view was taken from sections with radii between 9.6
and 12.0 nm. The arrows indicate the protofilament
axes. (Modification of figure by Amos and Klug, 1974,
with permission.)
microtubules spontaneously when certain
conditions are met. Weisenberg (1972) has
demonstrated that the in vitro assembly
process is temperature sensitive, requires a
nucleotide (usually GTP), and is dependent
upon protein concentration and upon pH
and ionic strength. The assembly reaction is
inhibited by Ca2+ at low concentrations (on
the order of 10~5 to 10~3M) and requires
Mg 2+ (Johnson and Borisy, 1974). At
higher concentrations, Mg2+ is also inhibitory. The effect of Ca2+ is dependent upon
the Mg2+ concentration. At low Mg2+ con-
FIG. 2. A model of the 3-dimensional reconstruction
of a singlet A-tubule showing the 4.0- and 8.0-nm
periodicities. The a and /3-tubulins are identified with
the 4.0 nm morphological units. (From Amos and
Klug, 1974, with the kind permission of the authors.)
652
JOSEPH BRYAN
FIG. 3. Distribution of proteins in an assembly competent tubulin preparation analyzed by SDS-gel electrophoresis. The tubulin was isolated from rabbit
brain by two polymerization-depolymerization cycles
using the procedures detailed by Shelanski et al.
(1973). The SDS-polyacrylamide gel conditions are
described in Bryan (1974). On the basis of staining, the
a//3 ratio is 1.05, the two MAP's are approximately
1.7:1 and account for approximately 7 to 8% of the
protein on the gel.
sembly of tubules in these preparations has
been followed by a variety of techniques
including centrifugation, viscosity (Borisy
et al., 1974), and light scattering (Gaskin et
al., 1975). Figure 4 illustrates the assembly
process using partially purified rabbit brain
tubulin and the light scattering assay. The
reaction is initiated by warming tubulin to
23°C in the presence of GTP, Mg2+, 4 M
glycerol, and MES buffer at pH 6.5. Assembly is monitored as a function of time by
measuring the increase in optical density at
653
CONTROLS OF MICROTUBULE ASSEMBLY
50
60
FIG. 4. Illustration of the light-scattering assay for
microtubule assembly (Gaskin et al., 1975). Assembly
was monitored by measuring the increase in optical
density at 350 nm as a function of time. The tubulin
preparation was isolated as described in Figure 2. The
solution conditions were: 0.1 M MES, 0.5 mM MgCh,
2mM GTP, and 4 M glycerol at pH 6.5. The reaction
was initiated by placing the samples at 23°C. The assembly reactions at various concentrations are illustrated. Samples taken for electron microscopy at various times during the reaction indicate formation of
microtubules.
350 nm. Plots of the extent of assembly as
measured by the plateau values versus the
concentration of added tubulin are shown
in Figure 5 for two temperatures. The data
are similar to those presented by Gaskin et
al. (1975) and Johnson and Borisy (1974),
and suggest that there is little assembly occurring until a critical concentration (Cc) is
exceeded. These results and the validity of
the light scattering assay have been verified
by assembling tubules at various protein
concentrations, collecting the tubules by
centrifugation, then directly measuring the
amounts of protein in the supernatant or
pellet. The results, shown in Figure 6, are in
direct agreement with those obtained using
the light scattering assay. A plot of the OD
increase at 350 nm (AOD350) versus the
amount of protein sedimented is linear
0.5 •
0.4
a
o
c
c
3
Maxim
20
30
40
Minutes at 23° C
(Fig. 7) in the concentration range studied.
This type of behavior, no assembly until
some critical concentration (approximately
0.25 mg/ml under these conditions) is
reached, agrees with the predicted characteristics for helical or tubular polymerization which have been explored by Oosawa
and his colleagues (Oosawa and Kasai,
1962; Oosawa and Higashi, 1967). Interpreted in terms of this theory, the data
argue that below 0.25 mg/ml the reaction
mixture consists primarily of tubulin dimers in equilibrium with a few small
polymers. Above this critical concentration,
a constant concentration of tubulin dimers
exists in equilibrium with long polymeric
microtubules. As the tubulin concentration
is increased, the amount of protein in microtubules increases, but the amount in dimers remains constant. The critical concentration corresponds to the point where
the chemical potential of a dimer in free
solution equals that of a dimer in the microtubule. The calculated equilibrium constant using 0.25 mg/ml is approximately 4.5
to 5.0 x 1 0 + 5 M - 1 . These characteristics of
tubulin assembly suggest a condensation
polymerization mechanism of assembly.
This would be analogous to the condensation of a liquid from a gas phase or the
crystallization of an inorganic salt from a
37°C yS
0.3
if
Jir
0.2
0.1
t
Cc
1
2
3
4
5
6
Initial Protein Concentration (mg/ml)
FIG. 5. A plot of the plateau or maximum optical
density at 350 nm versus the protein concentration of
the reaction mixture. The data shown for two temperatures extrapolate through a Cc = 0.25 mg/ml. The
solution conditions and the tubulin preparation are
those detailed in Figure 4.
654
JOSEPH BRYAN
protein sedimented at 100,000xg
V
6
Initial Protein Concentration
(mg/ml)
FIG. 6. Sedimentation analysis of microtubule assembly. Samples at various protein concentrations
were incubated for 30 min at 37°C, then centrifuged at
100,000 x g for 30 or 60 min at 25°C. The supernatants and pellets were analyzed for protein. The values
are either soluble protein (mg/ml) or protein sedimentable at 100,000 x g (mg/ml). The lines drawn
were fit by a least squares procedure, the indicated
Cc = 0.27 mg/ml. The data deviate from the predictions of Oosawa and his colleagues in that the concentration of soluble protein is somewhat larger than Cc
and tends to increase slightly as the protein concentration increases. Solution conditions and tubulin
preparation are those detailed in Figure 4.
saturated solution. Using the crystallization analogy, Oosawa and Higashi (1967)
have pointed out that the polymerization of
tubular polymers can be divided into two
stages, an initiation step and a growth phase.
The spontaneous formation of a tubule
must proceed through the initiation step,
which is usually rate limiting, then the
growth phase.
The problem of initiation or nucleation
of microtubule assembly can then be
considered at several levels. We must discriminate between nucleation by apparently non-tubulin structures like the
kinetochore (heterogeneous nucleation)
and nucleation or initiation by structures
which form spontaneously in tubulin solutions (homogeneous nucleation). Borisy
and Olmsted (1972) have described the
nucleated assembly of tubulin-containing
solutions and have shown that removal of
potential nuclei by high speed centrifugation blocked assembly. The characteristics
of these potential nuclei, called rings, have
been investigated in some detail by
Kirschner and his co-workers (1974). The
rings can be obtained by fractionation of
assembly-competent tubulin; they contain
a- and /3-tubulins and MAP's. There are, at
the moment, conflicting reports as to
whether the rings are in fact required for
spontaneous tubulin assembly. Kirschner
et al.' (1974) have reported that the ring
fraction, purified in the presence of Ca2+
will form microtubules if the Ca2+ is removed. Using the same procedures, the
dimer fraction will assemble rings if
glycerol is added to 4 M. Dentler et al. (1975)
and Murphy and Borisy (1974) have reported that dimers obtained by fractionation of cold depolymerized microtubules
will spontaneously assemble although no
rings are present. The latter results suggest
that tubulin solutions alone are capable of
Protein Sed. at 105G
(mg-ml)
FIG. 7. Comparison of sedimentation results with
light scattering results. Duplicate samples of tubulin at
various protein concentrations were either monitored
at 37°C by the light scattering assay or held for
sedimentation. After 30 min, samples were
sedimented at 100,000 g for 20 min at 25°C and the
proteins in pellets and supernatants analyzed. The
protein sedimented was plotted versus the increase in
optical density at 350 nm (AOD350) at the time of
initiation of sedimentation. The results are reasonably
linear over the concentration range used.
CONTROLS OF MICROTUBULE ASSEMBLY
self or "homogeneous" nucleation and subsequent assembly of microtubules.
EVIDENCE FOR NUCLEATION SITES
A variety of arguments have been used to
suggest that the kinetochores and centrioles or spindle poles organize the spindle
fiber microtubules. Mazia (1955) made the
suggestion on the basis of a differential solubility of the polar region of the isolated
MA. Inoue and Sato (1964) have termed
these structures "orienting centres" and
have given evidence that the birefringence
is stronger around these centers than in the
rest of the MA. Forer, using UV microbeam
techniques has provided further evidence
that the kinetochores are actually involved
in organizing chromosomal fibers (see
Forer, 1965, and for a more recent discussion, Forer, 1969, 1974). In addition, in the
electron microscope, microtubules are observed to insert directly into the
kinetochore (Harris, 1961, 1962).
Direct evidence that the kinetochores can
serve as sites for the initiation of tubulin
assembly into microtubules comes from the
recent work of Rebhun and his colleagues
(1974). They have isolated the MA from
Lytechinus using a procedure involving gentle lysis of the metaphase egg at room
temperature with a non-ionic detergent,
Triton X-100. The lysing medium contains
a calcium chelating agent (EGTA) and a
protease inhibitor and is buffered at a pH
and ionic strength known to support tubule
assembly in vitro. This procedure gives an
isolated spindle which is cold labile; if the
isolated spindle is cooled on ice, the birefringence drops. The MA remnant can now
be placed in solutions of partially purified
brain tubulin and warmed. This results in
the return of birefringence to essentially
control levels and distribution. A similar
series of experiments has been carried out
on isolated cells perfused with tubulin containing solutions (see Inoue et al., 1974, for
experiments with Chaetopterus, and Cande
et al., 1974, for experiments with cultured
mammalian cells). These experiments are a
direct demonstration that kinetochores and
poles, or perhaps kinetochores with some
residual tubulin attached, can nucleate the
655
assembly of heterologous tubulin.
A similar approach has been used by
Snell et al. (1974) to demonstrate that isolated basal bodies, near relatives of centrioles, from Chlamydomonas reinhardi can
serve as initiation centers for the assembly
of chick brain tubulin.
CALCIUM AS AN INTRACELLULAR REGULATOR OF
MICROTUBULE ASSEMBLY
The in vitro assembly experiments indicate that there are several potential regulators of the rate and extent of tubule assembly including nucleotides (Jacobs
1975), divalent cation concentration, and
protein phosphorylation (Rasmussen,
1970). Each of these has been suggested as a
possible regulator. The evidence for actual
regulation by any of these components is
meager and only for calcium is it sufficient
to merit discussion.
In vitro, Ca2+ will prevent spontaneous
assembly of tubules (Weisenberg, 1972;
Olmsted and Borisy, 1973) and will depolymerize existing microtubules (Gaskin
et al., 1975). The effective concentration of
Ca2+ is dependent upon the Mg2+ concentration; lower concentrations of Ca2+ are
more effective at higher Mg 2+ levels
(Rosenfeld and Weisenberg, 1974). The
kinetochore nucleated assembly of microtubules is sensitive to Ca2+; polymerization
is inhibited and existing tubules are depolymerized by 10~3M Ca2+ (at low Mg2+
levels) (Rebhun et al., 1974; Inoue et al.,
1974). In addition, the successful isolation
of the MA in the absence of supporting
glycols (Rebhun et al., 1975) requires the
removal of Ca2+ with chelating agents.
These in vitro experiments indicate that
microtubules are sensitive to calcium although the data do not yet conclusively indicate that this sensitivity occurs in a range
of physiological Mg2+ and Ca2+ concentrations. To verify that Ca2+ is a regulator, one
would like to be able to demonstrate, in
vivo, a fluctuation of Ca2+ ion throughout
the cell cycle with a marked decrease in
intracellular Ca 2+ during the formation
and assembly of the MA. At present there is
no direct evidence for such an intracellular
Ca2+ decrease during mitosis (cf. Baker and
656
JOSEPH BRYAN
Warner, 1974). There is indirect evidence,
however, which suggests that such a fluctuation may indeed occur. This comes from
the work of Petzelt (1972) characterizing
the activity of a Ca2+ activated ATPase
throughout the first few cell cycles in sea
urchin eggs: The enzyme is Ca2+ activated,
inhibited by Mg2+, Na+, and K+, and is
specific for ATP. Furthermore, the enzyme
is concentrated in the isolated MA (Maziaet
al., 1972). During the first cleavage divisions in 5. purpuratus, there are two peaks of
enzyme activity; one in interphase and a
second during metaphase. If puromycin is
added to eggs before fertilization, cell division is blocked (Hultin, 1961) although
DNA replication goes on (Wilt et al., 1967).
In eggs treated with puromycin, the Ca2+activated ATPase remains unchanged following a slight decrease at fertilization,
suggesting that the enzyme may be required for cell division. Using parthenogenetically activated eggs from Paracentrotus lividus, Petzelt and Ledebur-Villiger
(1973) have shown that the enzymatic activity increases when the MA or similar structures, monasters and cytasters, are formed.
Since chromosome movements do not go
on in the monasters and cytasters, Petzelt
and Ledebur-Villiger have argued that the
enzyme may function in the assembly of the
MA by acting to reduce the intracellular
Ca 2+ concentrations. The analog being
considered is a muscle cell in which a Ca2+activated ATPase in the sarcoplasmic reticulum is involved in regulation of the intracellular Ca 2+ concentration. This
suggestion is reinforced by the demonstration of Kinoshita and Yazaki (1967) that sea
urchin eggs possess a particulate system
which will sequester Ca2+, and by the report
of Cheney (1948) that caffeine, which
mediates Ca 2+ release from sarcoplasmic
reticulum (Weber and Herz, 1968; Weber,
1968), inhibits cell division in sea urchins.
Rebhun et al. (1975) have verified that caffeine (5 to 10 min) inhibits cell division in 5.
purpuratus and have reported that in Spisula
eggs caffeine treatment results in a loss of
birefringence in the meiotic spindle.
The notion that there is a vesicular system in the echinoderm MA for Ca 2+ uptake
is further reinforced by the morphological
observations of Harris (1961, 1962) who
demonstrated the presence of densely
packed membranous vesicles in the MA in
the region around the centrioles. These
appear to be derived from smooth endoplasmic reticulum and are obvious candidates for the site of the ATPase. There is an
unproven, but logical link between the
Ca2+-activated ATPase, the puromycin
sensitivity, and the extensive endoplasmic
reticulum in that Wilt et al. (1967) have
shown that a fraction containing the vesicles has a higher specific activity of labeling
with amino acids during the first cell cycle
than the other proteins of the MA. This
would be in line with the notion that the
ATPase is synthesized in a puromycinsensitive step early after fertilization, is
localized in the vesicles, and is required for
MA formation.
Following the line of argument that Ca2+
sequestering vesicles may be important in
tubulin assembly, we have looked at the
effects of several agents including caffeine,
Triton X-100, and phospholipase-A on the
assembly of microtubules in crude supernatants of chick embryo brain (Nagle and
Bryan, unpublished). These supernatants
contain a vesicle fraction which has been
reported to sequester Ca2+ (Nakamaru and
Schwartz, 1971), which can subsequently be
released by caffeine, Triton X-100, or
phospholipases (Nakamaru et al., 1967).
Addition of any one of the three agents
inhibits microtubule assembly in the crude
supernatants. Similar additions to partially
purified tubulin preparations, free of vesicles, has no effect on assembly. Furthermore, inhibition in crude supernatants can
be partially reversed with EGTA, suggesting Ca2+ release may be mediating the inhibition of assembly.
In summary, there is evidence that tubulin polymerization is inhibited by Ca2+ and
that pre-existing tubules are depolymerized if the Ca2+ concentration is
raised. There is further evidence which indicates that sea urchin eggs, and presumably other cells, have a vesicular system for
sequestering Ca 2+ . In the sea urchin, a
Ca2+-activated ATPase is closely correlated
with the appearance of the MA; if synthesis
of the enzyme is blocked using puromycin,
CONTROLS OF MICROTUBULE ASSEMBLY
no MA develops. The observed fluctuations
in the Ca2+-activated ATPase during the
cell cycle are consistent with a decrease in
intracellular Ca2+ at metaphase, but no actual fluctuation in intracellular Ca2+ has yet
been demonstrated. This problem has been
approached directly in Xenopus; the results
are consistent with a Ca2+ fluctuation but a
variety of other interpretations are possible
(Baker and Warner, 1974).
INHIBITORS OF SPONTANEOUS ASSEMBLY
Several lines of evidence directly support
the idea that cells capable of division have
the ability to suppress spontaneous assembly of microtubules. Burns and Starling
(1974) have reported that crude supernatants of sea urchin tubulin will not spontaneously assemble tubules. However, after
addition of microtubule "seeds," either
short pieces of sperm tail microtubules or
rat brain microtubules, further assembly
occurs by addition to the seeds. Similar results have been obtained in Spisula
homogenates by Weisenberg (1973); here
the "seed" appears to be an organizing
center which can be removed by low speed
centrifugation. On the other hand, if tubulin is removed from those cellular supernatants by purifying, for example, the cold
labile MA (Rebhun et al., 1974), then spontaneous reassembly can occur (Smith and
Rebhun, 1974). In short, the tubulin in
these supernatants is competent to assemble, but appears to be blocked from doing
so.
The question of tubulin concentration is
of some importance. Calculations using the
data of Burnside et al. (1973) for Arbacia
indicate that the amount of tubulin in these
eggs is sufficiently high (> 0.25 mg/ml) that
we would expect spontaneous assembly if
the supernatants behaved like brain preparations.
This failure of spontaneous assembly
seems to be a quite general phenomenon.
We have been unable to polymerize microtubules in CHO cell supernatants using a
variety of conditions (see also Rebhun et al.,
1975) and other cell lines including Hela
(Borisy, personal communication), neuroblastoma (Rosenbaum, personal communi-
657
cation), mouse L-cells (Petzelt and Bryan,
unpublished), and mouse ascites cells
(Donges and Paweletz, personal communication) all fail to assemble tubules under
conditions which are optimal for brain
tubulin assembly.
The reason for this failure, in at least the
sea urchin and two of the cell lines (CHO
and the ascites line), appears to be the presence of a heat-stable, nondialysable factor
which depresses spontaneous initiation of
assembly in these supernatants and can inhibit spontaneous assembly of partially purified brain tubulin. Results using sea urchin
factor are shown in Figure 8. At low concentrations of the factor, the effect is predominately on the initial rates of assembly;
at higher concentrations, the extent of assembly also drops. Figure 8 illustrates the
effect on the initial rates of assembly versus
the percentage of sea urchin cytoplasm
1.0
10
% of Sea Urchin Cytosol
FIG. 8. The effect of the sea urchin heat-stable factor
on rabbit brain tubulin assembly. Strongylocentrotus
purpuratus eggs were washed three times in filtered sea
water, homogenized in two volumes of 0.1 M MES, 0.5
mM MgCU at pH 6.5 and a 100,000g-60 min supernatant prepared. The supernatant was heated to 100°C
for 10 min in a boiling water bath and the 100,OOOg-60
min centrifugation repeated. The final supernatant
was dialyzed exhaustively against 0.1 M MES, 0.5 mM
MgCU at pH 6.5. The effect of the retentate was compared with an equal amount of the diffusate. The light
scattering assay was used to measure the initial assembly rates. The solution conditions are those given in
Figure 4. The data are given as the per cent of control
rate (no addition) versus the percentage of sea urchin
cytosol. The percentage of cytosol figures are normalized to the egg. A 1:50 dilution of the cytosol is
sufficient to produce a 50% decrease in the initial
assembly rate under these conditions using tubulin at
1.7 mg/ml.
658
JOSEPH BRYAN
added. The equivalent of 2% of the sea
urchin cytoplasm (a 1:50 dilution) reduces
the initial assembly rate by 50%. Several
trivial explanations for this inhibition including concentrations of divalent or
monovalent ions and increased protein
concentration have been ruled out. The
mechanism of inhibition is, however, not
yet clear. We have established that when
brain microtubules are dissolved in solutions containing the factor at concentrations sufficient to inhibit assembly, the formation of rings is greatly suppressed
(Nagle and Bryan, unpublished). Rebhun
et al. (1975) have also indicated that both
sea urchin and CHO cell supernatants are
devoid of rings. While these observations
are consistent with the idea that the rings
are the sites of initiation of spontaneous
assembly and that the factor inhibits spontaneous assembly by preventing ring formation, this argument is somewhat
weakened by the reports that rings are not
completely essential for tubulin polymerization. It remains to be demonstrated
whether the inhibitory factor acts on the
MAP's or perhaps by preferentially binding
to one of the configurations of tubulin
molecules which proceeds the actual initiation complex.
SUMMARY AND PROBLEMS
We have considered preliminary and
admittedly incomplete evidence that three
factors operate to control and order microtubule assembly during mitosis. These include: (i) the sites for nucleation or initiation of tubule assembly, the kinetochores
and centrioles or pericentriolar material;
(ii) a Ca2+-activated ATPase which may
control tubulin assembly-disassembly by
regulating the intracellular Ca2+ concentration; and (iii) a heat stable factor for suppressing spontaneous microtubule assembly. A schematic representation of how
these three factors could work is given in
Figure 9. Tubulin can polymerize into microtubules along one of two pathways.
"Homogeneous" nucleation can occur if
the tubulin concentration is above the critical concentration. This involves the formation of a suitable seed, perhaps the first turn
Homogenous
Nucleation
Randomly Oriented
Microtubules
Oriented
Chromosomal Fiber
Microtubule
FIG. 9. A hypothetical scheme for the control of microtubule assembly during mitosis in the sea urchin.
of the tubule helix or the lateral aggregation of 13 short protofilaments. This type of
assembly is believed to be blocked by the
heat-stable factor. The mechanism of this
block is unknown, but may involve either
specific binding to one of the intermediate
aggregation states of tubulin on the pathway to an active nucleus or "seed" or interaction with the MAP's. In situ this system
would operate to prevent random unoriented polymerization of microtubules
during mitosis and presumably at other
times in the cell cycle.
The second pathway is indicated as a
"heterogeneous" nucleation, implying that
the nucleation or initiation site either is not
composed of tubulin (reasonable for the
kinetochore) or is tubulin in a stable structure (the basal body case). It may be necessary to extend this definition to include
localized areas of elevated tubulin concentration such as the pericentriolar region
once their chemistry has been worked out.
These heterogeneous nuclei are unaffected
by the heat-stable factor. In the case of the
chromosome, for example, tubulin
polymerizes onto the kinetochore, forming
microtubules in an oriented and directed
fashion. During prophase and early
metaphase, the rate and extent of tubulin
polymerization onto a kinetochore may be
further regulated by a decreasing Ca2+
concentration as is suggested by the increased activity of the Ca 2+ -activated
ATPase. The increasing polymerization of
microtubules is reflected in the increased
birefringence observed in the polarization
microscope (see particularly Figure 7 in
Inoue and Sato, 1967, for data from
Lytechinus variegatus). In late anaphase and
telophase, the Ca2+-activated ATPase activity drops suggesting an increasing Ca 2+
CONTROLS OF MICROTUBULE ASSEMBLY
concentration. At this time the birefringence fades as microtubules are
depolymerized.
A variety of problems remain within this
hypothetical scheme. Little is known about
the chemistry of the heterogeneous nucleation sites and essentially no information is
available on the nucleation process itself.
The proposal that Ca2+ is, in fact, an intracellular regulator of tubulin assembly
remains in serious doubt despite the evidence presented here. Three crucial pieces
of evidence are missing. First, it is not at all
clear that the levels of free Mg2+ in a cell are
sufficiently high to insure that the tubulin
assembly reaction will be significantly affected by low Ca2+ levels. Measurements of
the free Mg2+ concentration in rat tissues
indicate that the levels in brain, kidney, and
liver are only on the order of 1 mM (Veloso
et al., 1973). At these Mg2+ concentrations,
inhibition of tubulin assembly in vitro requires Ca2+ concentrations in almost the
millimolar range.
While there is a large amount of Ca2+ in
echinoderm eggs, it is not yet clear that this
is ever free in the cytoplasm. Second, it is
not yet clear that Ca2+ can interact directly
with tubulin. The Ca2+ inhibition observed
in assembly-competent brain tubulin may
be an effect on the MAP's, which in turn
may not be present in non-brain cells.
Third, there is, as yet, no direct evidence
for a Ca2+ fluctuation correlated with microtubule assembly.
Finally, the nature and site of action of
the inhibitor(s) of spontaneous assembly
remain to be worked out.
A large number of problems remain in
this area and we can already anticipate that
echinoderms will be particularly useful in
their resolution.
REFERENCES
Amos, L. A., and A. Klug. 1974. Arrangement of
subunits in flagellar microtubules. J. Cell Sci.
14:523-549.
Bajer, A. 1973. Interaction of microtubules and the
mechanism of chromosome movement (zipper
hypothesis). 1. General principle. Cytobios 8:139160.
Baker, P. F., and A. E. Warner. 1972. Intracellular
calcium and cell cleavage in early embryos of
659
Xenopus laevis. J. Cell Biol. 53:579-581.
Borisy, G. G., and Olmsted, J. B. 1972. Nucleated
assembly of microtubules in porcine brain extracts.
Science 177:1196-1197.
Borisy, G. G., J. B. Olmsted, J. M. Marcum, and C.
Allen. 1974. Microtubule assembly in vitro. Fed.
Proc. 33:167-180.
Bryan, J. 1974. Biochemical properties of microtubules. Fed. Proc. 33:152-157.
Bryan, J., and L. Wilson. 1971. Are cytoplasmic microtubules heteropolymers? Proc. Nat. Acad. Sci.
U.S.A. 68:1762-1766.
Burns, R. G., and T. D. Pollard. 1974. A dynein-like
protein from brain. FEBS Lett. 40:274-280.
Burns, R. G.,and D. Starling. 1974. Them vitro assembly of tubulins from sea urchin eggs and rat brain;
use of heterologous seeds. J. Cell Sci. 14:411-419.
Burnside, B., C. Kozak, and F. C. Kafatos. 1973. Tubulin determination by an isotope dilution-vinblastine
precipitation method. The tubulin content of
Spisula eggs and embryos. J. Cell Biol. 59:755-762.
Cande, W. Z.,J. Snyder, D. Smith, K. Summers, and J.
R. Mclntosh. 1974. A functional mitotic spindle
prepared from mammalian cells in culture. Proc.
Nat. Acad. Sci. U.S.A. 71:1559-1563.
Cheney, R. H. 1948. Caffeine effects on fertilization
and development in Arbacia punctulata. Biol. Bull.
94:16-24.
Dentler, W. L., S. Granett, and J. C. Rosenbaum. 1975.
Morphological identification of the high molecular
weight proteins (MAPs) associated with in vitro assembled brain microtubules. J. Cell Biol. 65:237-241.
Erickson,. H. P. 1974. Microtubule surface lattice and
subunit structure and observations on reassembly. J.
Cell Biol. 60:153-167.
Erickson, H. P. 1975. The structure and assembly of
microtubules. In The biology of microtubules. New
York Academy of Sciences. (In press)
Forer, A. 1965. Local reduction of spindle fiber birefringence in living Nephrotoma suturalis (Loew)
spermatocytes induced by ultraviolet microbeam irradiation. J. Cell Biol. 25:95-117.
Forer, A. 1969. Chromosome movements during
cell-division, Pages 553-601 in A. Lima-de-Faria,
ed., Handbook of molecular cytology. North Holland Publishing Co.,
Forer, A. 1974. Possible roles of microtubules and
actin-like filaments during cell-division. Pages 319336 in G. M. Padilla, I. L. Cameron and A. M. Zimmerman, eds., Cell cycle controls. Academic Press,
New York.
Gaskin, F., C. R. Cantor, and M. L. Shelanski. 1975.
Biochemical studies on the in vitro assembly and
disassembly of microtubules. In The biology of microtubules. New York Academy of Sciences. (In
press)
Gaskin, F., S. B. Kramer, C. R. Cantor, R. Adelstein,
and M. L. Shelanski. 1974. A dynein-like protein
associated with neurotubules. FEBS Lett. 40:281286.
Harris, P. 1961. Electron microscope study of mitosis
in sea urchin blastomeres. J. Biophys. Biochem.
Cytol. 11:419-435.
Harris, P. 1962. Some structural and functional aspects of the mitotic apparatus in sea urchin embryos.
660
JOSEPH BRYAN
J. Cell Biol. 14:475-488.
puratus. Exp. Cell Res. 70:333-339.
Hultin, T. 1961. The effect of puromycin on protein Petzelt, C , and M. Ledebur-Villiger. 1973. Ca++metabolism and cell division in fertilized sea urchin
stimulated ATPase during the early development of
eggs. Experientia 17:410-411.
parthenogenetically activated eggs of the sea urchin
Paracmtratus lividus. Exp. Cell Res. 81:87-94.
Inoue, S., G. G. Borisy, and D. P. Kiehart. 1974.
Growth and lability of Chaetopterus oocyte mitotic Raff, R. A. 1975. Regulation of microtubule synthesis and utilization during early embryonic developspindles isolated in the presence of porcine brain
ment of the sea urchin. Amer. Zool. 15:661-678.
tubulin. J. Cell Biol. 62:175-184.
Inoue, S., and H. Sato. 1967. Cell motility by labile Rasmussen, H. 1970. Cell communication, calcium ion
and cyclic adenosine monophosphate. Science
association of molecules. J. Gen. Physiol. 50:259170:404-412.
288.
Jacobs, M. 1975. Tubulin nucleotide reactions and Rebhun, L. I., D. Jemiolo, N. Ivy, M. Mellon, and J.
their role in microtubule assembly and dissociation.
Nath. 1975. Regulation of the in vivo mitotic apIn The biology of microtubules. New York Academy
paratus by glycols and metabolic inhibitors. In The
of Sciences. (In press)
biology of microtubules. New York Academy of Sciences. (In press)
Johnson, K. A., and G. G. Borisy. 1974. Equilibrium
microtubule assembly: quantitation by sedimenta- Rebhun, L. I., J. Rosenbaum, P. Lefebvre, and G.
tion. J. Cell Biol. 63:314a.
Smith. 1974. Reversible restoration of the birefringence of cold-treated isolated mitotic apparatus of
Kinoshita, S., and I. Yazaki. 1967. The behaviour and
surf clam eggs with chick brain tubulin. Nature
localization of intracellular relaxing system during
(London) 249:113-115.
cleavage in the sea urchin egg. Exp. Cell Res.
47:449-458.
Rosenfeld, A., and R. Weisenberg. 1974. Role of Mg
Kirschner, M. W., R. C. Williams, M. Weingarten, and
and Ca in microtubule assembly. J. Cell Biol.
J. C. Gerhart. 1974. Microtubules from mammalian
63:578a.
brain: some properties of their depolymerization Sato, H. 1975. The mitotic spindle in aging gametes.
products and a proposed mechanism of assembly
Pages 19-49 in R. J. Blandau, ed., International
and disassembly. Proc. Nat. Acad. Sci. U.S.A.
Symposium, Seattle 1973. Karger, Basel.
Shelanski, M. L., F. Gaskin, and C. R. Cantor. 1973.
71:1159-1163.
Microtubule assembly in the absence of added nuLuduena, R., L. Wilson, and E. M. Shooter. 1974.
cleotides. Proc. Nat. Acad. Sci. U.S.A. 70:765-768.
Cross-linking of tubulin: evidence for the
Smith, G. W., and L. I. Rehbun. 1974. Isolation of the
heterodimer model. J. Cell Biol. 63:403a.
mitotic apparatus in a calcium-free medium. J. Cell
Mazia, D. 1955. The organization of the mitotic apBiol. 63:321a.
paratus. Symp. Soc. Exp. Biol. 9:335-340.
Mazia,D. 1961. Mitosis and physiology of cell division. Snell, W. J., W. L. Dentler, L. T. Haimo, L. I. Binder,
and J. L. Rosenbaum. 1974. Assembly of chick brain
Pages 77-412 in ]. Brachet and A. E. Mirsky, eds.,
tubulin on to isolated basal bodies ofChlamydomonas
The cell. Vol. 3."Academic Press, New York.
reinhardi. Science 185:357-359.
Mazia, D., C. Petzelt, R. O. Williams, and I. Meza.
1972. A Ca++-activated ATPase in the mitotic ap- Tilney, L. G., J. Bryan, D. Bush, K. Fujiwara, M.
Mooseker, D. B. Murphy, and D. H. Snyder. 1973.
paratus of the sea urchin egg, (isolated by a new
Evidence for 13 protofilaments: microtubules. J.
method). Exp. Cell Res. 70:325-332.
Cell Biol. 59:267-275.
Mclntosh, J. R., P. K. Hepler, and D. G. Van Wie.
1969. Model for mitosis. Nature (London) 224: Veloso, D., R. W. Guynn, M. Oskarsson, and R. L.
Veech. 1973. The concentrations of free and bound
659-663.
magnesium in rat tissues. J. Biol. Chem. 248:4811Murphy, D. B., and G. G. Borisy. 1974. The role of
4819.
tubulin-associated proteins in microtubule assembly. J. Cell Biol. 63:472a.
Warner, F. D., and I. Meza. 1974. Configuration of
flagellar microtubule subunits. J. Cell Sci. 15:495Nakamaru, Y., M. Kosakai, and K. Konishi. 1967.
512.
Some properties of brain microsome adenosine
triphosphatases activated by magnesium and cal- Warner, F. D., and P. Satir. 1973. The substructure of
cium. Arch. Biochem. Biophys. 120:15-21.
ciliary microtubules. J. Cell Sci. 12:313-326.
Nakamaru, Y., and A. Schwartz. 1971. Adenosine Weber, A. 1968. The mechanism of the action of caffeine on sarcoplasmic reticulum. J. Gen. Physiol.
triphosphate-dependent calcium binding vesicles;
52:760-772.
Mg++, Ca ++ adenosine triphosphatase and Na+, K+
adenosine triphosphatase: distributions in dog Weber, A., and R. Herz. 1968. The relationship between caffeine contracture of intact muscle and the
brain. Arch. Biochem. Biophys. 144:16-29.
effect of caffeine on reticulum. J. Gen. Physiol.
Olmsted, J. B., and G. G. Borisy. 1973. Characteriza52:750-759.
tion of micnotubule assembly in porcine brain exWeisenberg, R. C. 1972. Microtubule formation in
tracts by vistometry. Biochemistry 12:4282-4289.
vitro in solutions containing low calcium concentraOosawa, F., and S. Higashi. 1967. Statistical thertions. Science 177:1104-1105.
modynamics of polymerization and polymorphism
Weisenberg, R. C. 1973. Regulation of tubulin organiof protein. Progr. Theoret. Biol. 1:79-164.
zation during meiosis. Amer. Zool. 13:981-987.
Oosawa, F., and M. Kasai. 1962. A theory of linear and
helical aggregations of macromolecules. J. Mol. Wilt, F. H., H. Sakai, and D. Mazia. 1967. Old and new
protein in the formation of the mitotic apparatus in
Biol. 4:10-21.
cleaving sea urchin eggs. J. Mol. Biol. 27:1-7.
Petzelt, C. 1972. Ca++-activated ATPase during the
cell cycle of the sea Urchin Strongylocentrotus pur-