J. Cell Sci. 30, 331-352 (1978)
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THE ROLE OF SPINDLE POLE BODIES AND
MODIFIED MICROTUBULE ENDS IN THE
INITIATION OF MICROTUBULE ASSEMBLY
IN SACCHAROMYCES CEREVISIAE
BRECK BYERS, KATHLEEN SHRIVER
AND LORETTA GOETSCH
Departments of Genetics and Biochemistry,
University of Washington, Seattle, Washington, U.S.A.
SUMMARY
The spindle poles of the budding yeast, Saccharomyces cerevisiae, have been removed from
mitotic and meiotic cells by osmotic lysis of spheroplasts. The spindle pole bodies (SPBs)
- diskoidal structures also termed ' spindle plaques' - have been analysed for their ability to
potentiate the polymerization of microtubules in vitro.
Free SPBs were completely deprived of any detectable native microtubules by incubation
in the absence of added tubulin and were then challenged with chick neurotubulin, which had
been rendered partially defective in self-initiation of repolymerization. Electron microscopy
revealed that these SPBs served as foci for the initiation of microtubule polymerization in vitro.
Because the attached microtubules elongated linearly with time but did not increase in numbers
after the first stage of the reaction, it is apparent that there are a limited number of sites for initiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysis
of protein but not of DNA. The microtubule end proximal to the site of initiation on the SPB
is distinguished by a ' closed' appearance because of a terminal component which is continuous
with the microtubule wall, whereas the distal end has the 'open' appearance characteristic of
freely repolymerized neurotubules. SPBs which were partially purified on sucrose gradients
retained their ability to initiate the assembly of microtubules with the same structural differentiation of their ends. The occurrence of closed proximal ends on native yeast microtubules
suggests that closed ends may play a role in the initiation of microtubule polymerization in vivo,
as well as in vitro.
INTRODUCTION
The distribution and behaviour of microtubules suggests that these organelles play
an essential role in mitotic and meiotic division as well as in other changes of cellular
form (Porter, 1966). In order to understand the fundamental mechanisms responsible
for nuclear division, it is therefore reasonable to examine the manner by which the
functional arrangements of microtubules are determined. Morphological evidence has
suggested that microtubules arise at intracellular sites, which are believed to nucleate
the polymerization of the component protein precursor, tubulin (Tilney & Goddard,
1970). The behaviour of such sites, termed 'microtubule-organizing centres' by
Pickett-Heaps (1969), may therefore be pivotal to the control of microtubule-mediated
cellular processes.
Address correspondence to: Dr Breck Byers, Department of Genetics, SK-50, University
of Washington, Seattle, Washington 98195, U.S.A.
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B. Byers, K. Shriver and L. Goetsch
The development of conditions for the repolymerization of neurotubulin in vitro
(Weisenberg, 1972) has provided a method for the functional analysis of these centres
in vitro. Rebhun, Lefebvre & Rosenbaum (1973); Inoue, Borisy & Kiehart, (1974);
and Weisenberg & Rosenfeld (1975) have subsequently been able to demonstrate the
incorporation of exogenous neurotubulin into the organized structure of mitotic
and meiotic spindles. Moreover, it has been clearly demonstrated (Weisenberg, 1974;
Snyder & Mclntosh, 1975) that the augmented spindle includes microtubules which
have arisen entirely de novo at the spindle poles in the in vitro system. Further analysis
of the initiation process is hampered, however, by the fact that most initiating sites
within these spindles are indistinct and are diffusely arranged.
In certain eukaryotes, on the other hand, all of the microtubules radiate from precisely defined regions of densely staining amorphous material. This arrangement is
particularly evident in Saccharomyces cerevisiae (Robinow & Marak, 1966; Moens &
Rapport, 1971; Peterson & Ris, 1976), in which all of the microtubules end within the
discrete 'spindle plaques'. These diskoidal bodies, which are embedded in the nuclear
envelope throughout the life cycle, represent a type of spindle pole body (SPB) - the
organelle serving as the focus for microtubule arrangements in a wide range of lower
eukaryotes (see review by Kubai, 1975). Fundamental changes in the distribution of
microtubules, such as in the formation of the mitotic spindle, are necessarily
focused upon observable alterations of the SPBs to which they are attached (Byers &
Goetsch, 1975 a). In order to understand the mechanisms controlling microtubule
distribution, we have therefore undertaken an investigation of the manner in which
the SPBs mediate microtubule assembly. The present study has revealed that the
demonstrable initiation of neurotubulin polymerization in association with SPBs
in vitro results in a terminal modification of microtubule structure similar to that found
on native yeast microtubules.
MATERIALS AND METHODS
Strains and media
All of the experiments described here employed diploid strains of Saccharomyces cerevisiae.
Strain AP-i (Hopper & Hall, 1975) was used for tests of vegetatively-grown cells. Meiotic
stages were obtained with strain 212-1 (Simchen, 1974), which is homozygous for the temperature-sensitive allele cdc 4-1 (Hartwell, 1971) and may be arrested in meiosis at the double
SPB stage by elevation of the temperature to 33-5 °C (Byers & Goetsch, 19756). Vegetative
growth was maintained in YEPD medium consisting of 1 % Bacto-yeast extract, 2 % Bactopeptone, and 2 % dextrose. Presporulation medium PSP2 (Roth & Halvorson, 1969) consists
of 0-67 % yeast nitrogen base, 1 % yeast extract, 1 % potassium phthalate, and 1 % potassium
acetate supplemented with 40 fig/ml each of adenine and uracil. Sporulation medium SPM
consists of 0-3 % potassium acetate and 0-02 % raffinose.
Growth and induction of meiosis
Stationary phase stocks of strain 212-1 were maintained in medium YEPD. Cultures were
innoculated into presporulation medium PSP2 and maintained in logarithmic growth for 2
or more days before meiosis was induced by washing the cells twice with sterile water and
resuspending them in SPM at 22 °C (Roth & Halvorson, 1969). Electron microscopy of fixed
and embedded cells (Byers & Goetsch, 19756) demonstrated that the majority of cells were
at the single SPB stage at 2 h after the transfer and at the double SPB stage at 6 h.
Yeast spindle pole bodies
333
Spheroplasting and lysis
Spheroplasts were prepared by methods derived from the procedure of Peterson, Gray &
Ris (1972). Cells were washed with sterile water, resuspended in a pretreatment medium consisting of i M NaCl, 0-02 M ethylenediamine tetraacetic acid (EDTA), o-i M /?-mercaptoethanol,
and 0-2 M tris-hydroxymethylaminomethane-HCl (Tris-HCl), pH 9-0, and were incubated for
10 min at 22 CC. The cells were then transferred to the spheroplasting medium, consisting of
1 M NaCl, 0012 M sodium phosphate-citrate buffer, pH 5-8, and 10% (v/v) glusulase (Endo
Laboratories, Inc., Los Angles, California). After incubation for 30 min at 22 °C, the spheroplasts were washed twice by pelleting and resuspension in spheroplast stabilization buffer,
consisting of 0-7 M sorbitol and 0-012 M sodium phosphate-citrate buffer, pH 5-8. Lysis of the
spheroplasts was accomplished by resuspending the washed pellet in microtubule polymerization buffer, MPB (see below). This also resulted in lysis of the nuclei, freeing the spindles
or spindle poles in the lysate.
Chick neurotubulin extracts
Neurotubulin was prepared from the brains of 20-day chick embryos by the thermal
cycling method of Weisenberg (1972), as modified by the addition of glycerol (Shelanski,
Gaskin & Cantor, 1973). The brains were Dounce homogenized in microtubule polymerization
buffer (MPB) consisting of o-i M 2(i\T-morpholino)ethane sulphonic acid (MES), pH 6-4, 1 mM
ethyleneglycol-ta's(/?-aminoethylether^ATAT'-tetraacetic acid (EGTA),0-5 mM MgCU and 2 mM
guanosine triphosphate (GTP). The homogenate was chilled to 4 °C and centrifuged at 130000 g
for 60 min in a Spinco 6oTi rotor. The supernatant was diluted with an equal volume of 25 %
glycerol in MPB and incubated 30 min at 37 °C before a second 130000 g centrifugation for
60 min at 25 °C. Tubulin pellets were quickly frozen by immersion in liquid nitrogen and were
stored at —70 °C. Tubulin was resuspended at 5 mg/ml from thawed pellets by Dounce
homogenization in one-fourth the original volume of MPB at 4 °C and centrifuged at 4 °C
for 90 min at 230000 g to yield a 'high-speed' supernatant deficient in the potential for selfinitiation of microtubule polymerization. For some experiments, a 'low-speed supernatant'
was obtained from the original thawed pellets by centrifugation instead at 130000 g for 60 min
at 4 °C. One-fourth volume of 100% glycerol was added to the tubulin supernatants before
polymerization tests.
Polymerization of neurotubulin on spindle pole bodies
The assembly of microtubules in association with free SPBs was assayed by either of 2
procedures. In procedure A, the lysate was applied to Formvar-coated grids (80 bars per cm),
which were rinsed in MPB and transferred to the surface of a drop of neurotubulin solution.
Incubation of this preparation at either 20 or 37 °C permitted polymerization of microtubules
in association with the SPBs adherent to the Formvarfilm.In procedure B, the lysate was directly
mixed with the neurotubulin solution; this mixture was incubated at 20 or 37 °C and subsequently sampled by touching it with Formvar-coated grids. In either procedure, the samples
were then rinsed twice with distilled water and negatively stained by touching the grid to a
drop of 1 % uranyl acetate and blotting it dry. Partial disruption of the microtubules from their
association with the SPBs was sometimes enhanced by soaking the sample in the negative
stain for 5 min before blotting dry. This disruption facilitated observation of the microtubule
ends which had been attached to the SPB and had therefore been difficult to observe in the thicker
stain immediately surrounding the SPB.
Enzymic hydrolysis
The sensitivity of the initiation potential to enzymic hydrolysis was assayed by a modification
of procedure B; selected enzymes were added to the yeast lysate in MPB and the mixture was
preincubated 30 min at 30 °C before addition of neurotubulin. Deoxyribonuclease I and ribonuclease A (Worthington) were used atfinalconcentrations of 50 /*g/ml and 1 o/tg/ml, respectively.
The efficacy of these nuclease treatments was determined by electron microscopy (see Results).
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CEL
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B. Byers, K. Shriver and L. Goetsch
Trypsin (Sigma, type V) was used at 10 /tg/ml and its activity was arrested at the end of the
preincubation by the addition of 20 /tg/ml soybean trypsin inhibitor (Sigma, type II-S). The
proteolytic activity of the trypsin in MPB was assayed by the spectrophotometric analysis of
a-JV-benzoyl-AT-arginine ethyl ester HC1 (Sigma). Under these buffer conditions, the trypsin
treatment represented 15 BAEE units of activity (14% of the standard activity at pH 7-6) and
was reduced 3-fold by the addition of trypsin inhibitor.
Density gradient centrifugation of plaques
Spheroplasts were lysed by dilution into MPB, and the lysate was incubated for 30 min at
37 °C in 0-05 mg/ml deoxyribonuclease I before being layered on to a continuous gradient of
0-3 to i-8 M sucrose in MPB. Following centrifugation at 30000 rev/min at 4 °C for 30 min
in a Spinco SW41 rotor, the gradients were fractionated by pumping the solution from the
bottom. Aliquots of the fractions were analysed for their optical density at 230 nm to detect
components of the lysate and by optical refraction to characterize the gradient of sucrose
density. Other aliquots were tested for their content of SPBs competent to induce microtubule
polymerization by applying a droplet to a grid and inverting the grid on a droplet of highspeed supernatant tubulin for a 10-min incubation at 37 °C. The grids were then negatively
stained with 1 % uranyl acetate and the number of microtubule-associated SPBs per 10 grid
squares was determined by electron microscopy.
RESULTS
Electron microscopy of lysates
Spindle pole bodies (SPBs) were easily identified in negatively-stained lysates of
spheroplasts prepared from vegetative yeast cultures, confirming earlier observations
of Borisy, Peterson, Hyams & Ris (1975) and Peterson & Ris (1976). If spheroplasts
were lysed directly on the water surface to which electron-microscope grids were then
applied, the SPBs were almost invariably found in the vicinity of the released chromatin. Microtubules were found to remain attached to the SPBs in the same configuration as that seen in intact cells by thin-section electron microscopy. Lysates of
actively growing vegetative cultures included identifiable single SPBs, double SPBs
(which had duplicated but not undergone separation), and complete spindles about
1 /tm long. Longer spindles, known from thin-section electron microscopy to arise
late in nuclear division, were rarely seen; this stage therefore appears to undergo
selective loss during spheroplasting (Peterson & Ris, 1976).
Lysates of spheroplasts prepared from early meiotic stages gave a better yield of
interpretable preparations than those from vegetative cells because the chromatin
was less adherent and rarely obscured the microtubules. Stages similar to those in
the vegetative cells - single SPBs, double SPBs (Fig. 1 A, C), and complete spindles were found in the sequence of stages leading up to meiosis I (Moens & Rapport,
Fig. 1. Spindle pole bodies (spb) released by osmotic lysis of spheroplasted meiotic
cells (strain 212-1 at double SPB stage after 6 h in meiosis). A, negative staining immediately after lysis reveals microtubules with closed ends (ce) proximal to the double
SPB (one arrow to each element) and open ends (pe) located distally. B, greater disruption of microtubule-SPB attachment reveals several closed ends (ce) adjacent to the
SPB. C, microtubule-free double SPB after 15 min incubation in MPB before staining,
x 65000.
Yeast spindle pole bodies
335
pb
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B. Byers, K. Shriver and L. Goetsch
2A
B
Fig. 2. Pairs of SPBs freed by lysis of cells in meiosis I (at 8 h in meiosis). A, SPBs
freed from a single spindle by incubation in MPB to remove native microtubules.
x 80000. B, microtubule addition to same preparation after incubation with neurotubulin. x 15000. spb, spindle pole body; v, vesicles.
Yeast spindle pole bodies
337
1971). The SPBs measure about 50 nm in the dimension parallel with the attached
microtubules and 300 nm in the dimension perpendicular to the microtubules. These
and other structural features are consistent with the morphology of these bodies as
seen in thin sections. In addition, however, the negatively-stained preparations revealed a previously undetected modification of mierotubule structure. This modification was revealed by the difference in the distribution of the negative stain at the
2 ends of each mierotubule (Fig. 1 A, B). The end distal to the SPB has the appearance
typical of microtubules prepared from neurotubulin alone, the stain being uniformly
distributed in the axial region and occasionally revealing the frayed protofilaments.
This distribution of the stain indicates that the distal mierotubule end may be defined
as the end of an open cylinder, there being no interruption at the end of the mierotubule between the lumen and the exterior. We will henceforth designate ends of this
appearance as'open'. The end attached to the SPB, on the other hand, appears to lack
any opening to the exterior but instead consists of a closed surface which is continuous
with the mierotubule wall and excludes the stain to the same extent. We will designate
this end as 'closed'. By analogy, the yeast spindle mierotubule could be likened to a
rimless test tube, the open top of the test tube simulating the open end distal to the
SPB and its closed bottom simulating the closed proximal end of the mierotubule. The
closed end of the mierotubule sometimes displays the even curvature seen in the test
tube bottom, but is often more angular in profile (as may be noted in some views in
Fig. IB).
The polarity of these microtubules with respect to their SPB attachment was derived
from 2 sorts of observations. In the first place, observations of intact spindle poles
showed that the proximal ends were usually embedded so deeply in the negative stain
surrounding the SPB that their form could not be seen. Those few which could be
seen were all of the closed form, whereas the distal ends were easily seen and were
invariably open. Secondly, the occasional partial disruption of spindle poles permitted
unambiguous observation of the proximal ends of the microtubules. When the attachment between the proximal end and the SPB was disrupted, the proximal end
was clearly seen in negative stain of the same depth as that surrounding the distal end.
A few microtubules (about 10 % of the unattached ones) appeared to have been derived
by breakage because they had 2 open ends, one of which was adjacent to the open end
of another mierotubule. The remaining majority of well stained microtubules were
seen to have one closed end, which was almost always nearer the SPB than was the
open end. The minor fraction (less than 5%) of the opposite orientation must have
been inverted upon disruption because the distal ends of attached microtubules
were never (in 220 unambiguous observations) of the closed form. These observations
therefore indicate that the proximal end bears a distinct structural modification.
Whether this modification is associated with attachment to the SPB or with the initiation of polymerization at this site will be considered later.
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B. Byers, K. Shriver and L. Goetsch
Depolymerization and repolymerization of microtubules on spindle pole bodies in vitro
In order to examine the suspected control of microtubule polymerization by SPBs,
conditions were established for affecting the loss and reformation of microtubules in
cell-free lysates. Upon examining the depolymerization of native yeast microtubules,
we found that lysis of cells in microtubule buffer (Weisenberg, 1972) followed by
incubation at 37 °C for 5 min or longer resulted in the complete loss of any microtubules detectable by electron microscopy (Figs. 1 c, 2A). This loss made the SPBs
difficult to detect with certainty, but certain structural features permitted their
identification. Bodies of the appropriate form and dimensions were frequently seen
in the vicinity of the chromatin, where microtubule-laden SPBs would be found in
unincubated samples. Moreover, a single SPB could often be identified by the characteristic folded trilaminar membrane comprising the 'half-bridge' (Byers & Goetsch,
1974) along one margin of the main SPB. Double SPBs were unambiguously identified
(Fig. 1 c); some showed the intervening complete 'bridge' of folded unit membrane.
These free SPBs were frequently also characterized by their spatial association with
circular vesicles, 28-30 nm in diameter, which were clearly revealed by negative
staining. This association is discussed more fully later.
The ability of these microtubule-free SPBs to initiate the polymerization of tubulin
was then tested. Lacking a system of yeast tubulin competent to repolymerize in
vitro, we used chick neurotubulin with a high potential for repolymerization. After
one cycle of polymerization in a low-speed supernatant, the microtubules were
depolymerized in the cold and the tubulin was subjected to more extensive ultracentrifugation in order to reduce its potential for self-initiation of polymerization
(Borisy & Olmsted, 1972). This tubulin was then combined with lysates containing
microtubule-free spindle pole bodies either after adhesion of the SPBs to Formvar
films on electron-microscope grids (procedure A) or in free suspension (procedure B). In
either case, electron microscopy demonstrated that incubation at 37 °C resulted in the
polymerization of microtubules in association with the SPBs (Figs. 2B, 3).
Initial experiments on total yeast lysates were difficult to interpret because the
microtubules were often entangled in the masses of chromatin adjacent to the SPBs.
It was unclear whether this entanglement represented (1) some initiation of polymerization within the chromatin (perhaps at kinetochores), (2) the specific adhesion
of microtubules to chromatin, or (3) non-specific tangling. In any case, the confusion
of chromatin-associated microtubules was eliminated in subsequent experiments by
adding deoxyribonuclease to the preincubation mixture used to depolymerize native
microtubules. This treatment removed most visible chromatin from the Formvar
films but did not hinder the subsequent initiation of microtubule polymerization on
the SPBs. In these preparations, the vast majority of microtubules could easily be seen
to arise from the SPBs. The number of microtubules in the background (unassociated
with the SPBs) varied with the tubulin preparation but was usually less than 10 per
grid square (2500/«m2). In a typical preparation, the material released from two to
four spheroplasts was seen within one grid square. Each area of lysis contained one
duplicated SPB with 20-30 microtubules radiating from each half of the double SPB.
Yeast spindle pole bodies
339
B
Fig. 3. Time course of neurotubulin polymerization in vitro on to SPBs (after depolymerization of native microtubules as in Fig. 1 c) of 6 h meiotic cells, A, B, C, 5,
10 and 25 min, respectively. All x 20000.
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B. Byers, K. Shriver and L. Goetsch
Therefore, over 90% of the repolymerized microtubules seen were associated with
the identifiable SPBs.
Control experiments established that microtubule formation in this mixed system
was subject to constraints similar to those of a simple neurotubulin repolymerization
system. No microtubules appeared in normal preparations maintained at o °C, nor
did microtubules form if o-i mM colchicine was added to the preparation before incubation at 37 °C. Therefore, the in vitro system has the same sensitivity to cold and
colchicine as does the neurotubulin system alone, although yeast microtubules persist
at this concentration of colchicine in vivo (Haber, Peloquin, Halvorson & Borisy,
1972).
40
Time, min
Fig. 4. Microtubule number (upper panel) and microtubule length (lower panel) in
the time course of neurotubulin incubation in vitro with mixed single and double
spindle pole bodies (from lysed spheroplasts of strain 212-1 after 6 h in meiosis)
preincubated in MPB to remove native microtubules. Each time point represents
measurements on 20 SPBs, and vertical bars indicate the limits of one standard
deviation for these data.
Properties of microtubule repolymerization
In order to determine the role of the spindle pole bodies in the reformation of microtubules in vitro we then analysed the kinetics of the process in DNase-treated lysates.
A lysate containing SPBs devoid of microtubules was mixed with high-speed supernatant neurotubulin and incubated at 37 °C. Samples were taken by touching grids
to the mixture at successive times of incubation; the grids were then stained for
Yeast spindle pole bodies
341
electron microscopy. This time course ofrepolymerization demonstrated that initiation
was rapid and that the outgrowth of the microtubules was linear with time (Fig. 3).
The linearity of this outgrowth clearly establishes that the microtubules seen are not
remnants of the native spindle but are newly initiated in vitro. Furthermore, the overall length of the microtubules eventually formed (Fig. 4) far exceeds the length of
the native microtubules originally present in the yeast cell. It is therefore obvious
that at least the majority of each microtubule was formed in vitro.
The same samples were also used to determine the increase in microtubule numbers
emanating from each SPB during the incubation. Because unfavourable orientation
or staining often prevented our determining whether or not an individual SPB had
undergone duplication (in preparation for meiosis I), we simply counted the microtubules per focus of polymerization, whether it represented a single or a double SPB.
The data (Fig. 4) demonstrate that the microtubule number did not increase significantly after the first 5-10 min of incubation, but persisted at a stable value as the
microtubules continued to elongate. One must conclude, therefore, that the potential
for initiation of microtubule assembly had become saturated in spite of adequate
neurotubulin for extensive further elongation. These data also show that it is unlikely
that microtubules are simply aggregating on the SPBs rather than being initiated
there. If aggregation were occurring, it would have had to undergo completion within
the first few minutes of incubation.
Macromolecular nature of the initiating sites
After finding that spindle pole bodies devoid of visible microtubules could initiate
neurotubulin polymerization, we attempted to determine what macromolecular components of the SPBs were essential to the initiation process. We had already found that
deoxyribonuclease treatments sufficient to disrupt all visible chromatin did not prevent
initiation of polymerization. Similar tests with ribonuclease A were complicated by
the fact that this enzyme may have a stimulatory effect on neurotubulin repolymerization (Bryan, Nagle & Doenges, 1975). The treatments employed (see Methods),
which were sufficient to eliminate all visible ribosomes from electron-microscopic
preparations of the lysate, appeared to stimulate the formation of microtubules in
association with the SPBs, but the number of unattached (background) microtubules
was also increased to such an extent that the apparent effect of ribonuclease on SPBassociated polymerization remained inconclusive.
The requirement for trypsin-sensitive components was, on the other hand, clearly
demonstrable. Initial tests with 15 BAEE trypsin units per ml during preincubation
and incubation with tubulin resulted in a failure to form microtubules, probably
because of the direct effect of trypsin on the tubulin itself. This effect was subsequently avoided by adding soybean trypsin inhibitor after preincubation in trypsin,
but before the addition of tubulin. Under these conditions, the usual numbers of free
microtubules were seen to have arisen in the background but none was found attached
to the SPBs, thereby demonstrating the trypsin-sensitivity of their potential for initiation. The absence of any inhibitory effect by trypsin and trypsin inhibitor together
was proven by the substantial initiation of microtubules if both trypsin and inhibitor
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B. Byers, K. Shriver and L. Goetsch
Fig. S- Differentiation of ends of microtubules assembled in vitro (as in Fig. 3) for 15
min. A, prolonged uranyl acetate negative stain has dissociated several microtubules
from the SPB. x 24000. B, detailed view of proximal closed ends similar to those of
native microtubules. x 70000. c, detailed view of distal open ends, x 70000. ce,
closed end; oe, open end; v, vesicles.
Yeast spindle pole bodies
343
were present throughout both the preincubation and the subsequent incubation with
neurotubulin. We conclude that the potential to initiate polymerization is sensitive to
trypsin and insensitive to deoxyribonuclease as well as, perhaps, to ribonuclease.
These results suggest that sites responsible for the initiation of polymerization depend upon the integrity of proteinaceous components.
Anatomy of the repolymerized microtubules
The microtubules induced to assemble in vitro were similar in appearance to the
native yeast microtubules originally seen in simple lysates. Even when the lysate was
preincubated in the absence of neurotubulin, so that no remnants of native microtubules were detectable by electron microscopy, the microtubules which formed
upon subsequent addition of neurotubulin were closed at the proximal end. Although
the microtubule ends attached to the SPB were rarely detectable in the dense negative
stain in this region, all proximal ends seen were of the closed form. As in the case of
native microtubules, the proximal ends were more clearly seen if they had become
detached from the SPB during negative staining (Fig. 5). Detached microtubules
were found to have only one closed end, which usually remained nearer the detached
SPB. It is probable that the occasional microtubule with its closed end distal to the
SPB had become inverted from its original orientation, because the distal ends of the
reassembled microtubules which had remained attached were invariably of the open
form. These distal ends were frequently also splayed, revealing separated protofilaments
or tufts of helical material similar to that generally seen during microtubule assembly
(Kirschner & Williams, 1974).
Banding of spindle pole bodies on sucrose gradients
The preceding experiments demonstrate that SPBs freed by lysis of spheroplasts
remain as discrete bodies which will, when challenged with neurotubulin competent
to repolymerize, stimulate the assembly of microtubules. In order to establish whether
this stimulation of assembly depends upon functions residing within the SPBs alone,
gradient separation of these bodies from soluble components of the lysate was undertaken.
Spheroplasts were lysed by dilution in MPB with no added sorbitol and were layered
on to sucrose gradients. Initial attempts at the centrifugal separation of lysate components were hindered by the fact that the DNA freed from lysed nuclei made a viscous
layer which trapped other components. Because the potential for initiation of microtubule assembly was insensitive to deoxyribonuclease, subsequent samples were treated
with this enzyme as described. Following centrifugation, fractions of the gradients
were touched to grids, which were in turn applied to high-speed supernatant tubulin
in order to induce the assembly of microtubules. Negative staining and electron microscopy then revealed the distribution of SPBs among the fractions.
In earlier experiments on simple lysates, a centre from which microtubules radiated
could be identified as an SPB not only by its anatomy but because of its presence
within the localized debris released by lysis of a single spheroplast. Because this debris
was dispersed and partially removed during the gradient procedure, the identification
B. Byers, K. Shriver and L. Goetsch
344
i,
ce
Fig. 6. Microtubules polymerized in association with gradient-separated SPB
fractions. A, pairs of SPBs (double arrows) released from strain 501 iD cells after 3 h of
arrest of vegetative growth at 36 °C. Note low level of free microtubules in background,
x 10000. B, remnants of 2 SPBs from fraction 6 of the gradient shown in Fig. 7. ce,
closed end; v, vesicles, x 80000.
Yeast spindle pole bodies
345
of centres of radiating microtubules as SPBs relied solely on their anatomy. We did
find, in fact, that we could often identify the 2 components of the duplicated SPB if
the gradient had been prepared from spheroplasts of meiotic cells at this stage, but
unfavourable staining or angle of view frequently obscured observations to this
resolution. On the other hand, this ambiguity was not encountered in the analysis of
gradients of lysates of strain 5011D (temperature-sensitive for cdc 24) after temperature
arrest of vegetative growth. Spheroplasts of these cells regularly release large numbers
of intact complete spindles; their microtubules rapidly undergo depolymerization but
10
Fraction
15
Fig. 7. Isopycnic sucrose gradient separation of double SPB fraction from other components of lysed spheroplasts prepared after 6 h in meiosis (see Methods). The bottom
of the gradient is at the left. Application of droplets of fractions to grids followed by
challenge with neurotubulin reveals a maximum concentration of free SPBs (closed
circles in upper panel) in fraction 6 (146 M sucrose), whereas the bulk of debris indicated by light scattering (O.D.230 nm, open circles) is maximal in fraction 7 (1-40 M
sucrose). Low molecular weight components remain in fractions 15-20 at the top of the
gradient. A> sucrose concentration.
their SPBs remain attached to one another in pairs, apparently interconnected by
remnants of the nuclear envelope or nuclear cortex. Challenge of these lysates with
tubulin results in the appearance of distinct pairs of centres from which microtubules
radiate (Fig. 6A). The 2 members of each pair lie less than 1-5 /«m apart (more than
100 observations) whereas each pair is usually separated from the next by more than
8 /mi. This decidely non-random distribution of the centres is consistent with the
expected pairing of SPBs, further establishing the identity of these centres of assembly
with the SPBs themselves.
Whether from meiotic or vegetative cultures, the SPBs were consistently found to
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B. Byers, K. Shriver and L. Goetsch
Yeast spindle pole bodies
347
form a band at an apparent density slightly greater than the peak of light-scattering
material (Fig. 7). These partially purified SPBs retained the ability to induce microtubule assembly as rapidly as those recovered directly from unseparated lysates.
Moreover, these microtubules again bore a closed end adjacent to the site of initiation
at the SPB (Fig. 8). Although the current procedures do not yet permit a more thorough
purification, this partial separation from other components - particularly from the low
molecular weight components remaining at the top of the gradient — indicates that the
induction of microtubule assembly by a mechanism producing a closed proximal end
resides principally in the spindle pole bodies alone.
Vesicles associated with spindle pole bodies
SPBs freed from lysed spheroplasts were usually found associated with distinct
circular vesicles, 28-30 nm in diameter (Fig. 2A). From 0 to 12 of these vesicles were
found near each SPB, but they were only rarely seen at greater distances. They were
occasionally found adjacent to SPBs which retained their native microtubules and
were often seen in association with those upon which neurotubulin had been polymerized (Fig. 5B). They were also found in association with SPBs subjected to sucrose
gradient centrifugation (Fig. 6 B), demonstrating the persistence of their association
with the SPBs under these conditions in vitro. The role of these vesicles is not evident
from the present observations.
DISCUSSION
Every microtubule in Saccharomyces cerevisiae appears to have at least one of its
ends associated with the spindle pole bodies (SPBs) during both mitosis (Robinow &
Marak, 1966) and meiosis (Moens & Rapport, 1971). In the present study, we have
investigated the mechanisms which may be responsible for the generation of this
arrangement of microtubules. Negative staining of SPBs freed from spheroplasts by
osmotic lysis has revealed a structural differentiation of the end attached to the SPB.
Unlike the distal end, which appears to be a frayed open cylinder, the proximal end
is found to consist of a closed hemispherical, or sometimes angular, surface continuous
with the microtubule wall. We have designated the former type of ends as open and
the latter as closed.
Incubation of the lysate in microtubule assembly buffer resulted in the disassembly
of the native microtubules. Subsequent challenge of the lysate with avian neurotubulin
resulted in the initiation of microtubule assembly in vitro in association with the SPBs,
thereby demonstrating that these bodies can, indeed, serve as centres for the initiation
of microtubule assembly. This result confirms a preliminary report by Borisy et al.
(1974) of neurotubulin assembly on SPBs freed from vegetative yeast. In the present
Fig. 8. Microtubules polymerized in. association with gradient-separated SPBs from
meiotic cells (as in Fig. 6B). A, attached microtubules have open ends distally (open
arrows), x 90000. B-F the closed ends (solid arrows) of these microtubules disrupted
from SPBs were nearer the SPB than the open ends (open arrows), x 110000.
348
B. Byers, K. Shriver and L. Goetsch
study, dealing principally with meiotic cells, we have found in addition that the proximal ends of microtubules formed in vitro display a closed configuration similar to
that present in native yeast microtubules. Moreover, we have noted that the number
of microtubules arising on each SPB in vitro achieves saturation early in the reaction
in spite of continued elongation of the microtubules present. This saturation
phenomenon suggests that each SPB may bear a limited number of discrete sites for the
initiation of microtubule assembly. Each such site may then provide for the initiation of a
single microtubule with a closed end adjacent to the SPB, thereby accounting for
the fixed number of native spindle microtubules found by electron microscopy of
whole cells (Peterson & Ris, 1976).
We have also noted that spherical vesicles, 28-30 nm diameter, are found associated
with the spindle pole bodies upon microtubule depolymerization. These are similar
in appearance to vesicles frequently seen in association with SPBs in thin sections of
whole cells (unpublished observations). Their association with a site for the initiation
of microtubule assembly also suggests some similarity to large vesicles recently noted
near the spindle poles of mammalian cells (Gould & Borisy, 1976) but it remains
unknown whether they play any role in the initiation process.
Sites controlling microtubule assembly
In a wide variety of cellular forms, microtubules appear to arise from zones of
densely staining amorphous material (Tilney, 1971) similar in appearance to that
comprising the spindle pole bodies. For example, Brown & Bouck (1973) have demonstrated that the extensive arrays of microtubules in theflagellateOchromonas terminate
in association with dense material of this type. The initiation of microtubule repolymerization following transient depolymerization in vivo appears to occur at these sites.
Stearns, Connolly & Brown (1976) have more recently described similar material in
association with the basal bodies of the alga Polytomella agilis and have further demonstrated the ability of this material to potentiate the initiation of neurotubulin polymerization in vitro. These observations suggest that the initiating function serves as
the mechanism controlling microtubule distribution both in vivo and in vitro. If
microtubule distribution is, indeed, determined by initiating sites, then the fundamental basis for control must be sought in the processes which redistribute and
regulate the activity of these sites. One may then envisage that the varied sites controlling microtubule polymerization undergo reorganization in the course of the cell
cycle. During mitosis in animal cells, for example, some sites are discretely localized
at the kinetochores (Telzer, Moses & Rosenbaum, 1975), whereas many sites must
occur in the region immediately surrounding the centrioles. During interphase, on
the other hand, the controlling sites must reside in more widely dispersed regions of
the cell. Direct observations on the behaviour of the 'pericentriolar satellites' during
mitosis of HeLa cells demonstrates, in fact, that these structures, which are clearly
associated with the ends of some of the microtubules, undergo sequential changes in
their distribution (Robbins, Jentzsch & Micali, 1968). The precise analysis of this
redistribution is hampered, however, by the indistinct morphology and inadequately
defined staining properties of the satellites.
Yeast spindle pole bodies
349
By contrast, all apparent centres for microtubule organization in S. cerevisiae are
clearly defined throughout the entire life cycle. Every microtubule in the cell ends at
a discrete spindle pole body. Therefore, any redistribution of microtubules is necessarily accompanied by changes in these readily observable sites (Byers & Goetsch,
1974). During vegetative growth, for instance, the single SPB duplicates in coincidence
with bud emergence and cytoplasmic microtubules extend from the outer surface of
the duplicated SPB to the base of the emerging bud. Intranuclear microtubules,
which extend inward from the opposite side of the SPB, end within the interior of
the nucleus until the mitotic spindle forms between the separating SPBs. The terminal
phenotypes of several temperature-sensitive cell division cycle mutants isolated by
Hartwell, Culotti & Reid (1970) demonstrate that specific changes in SPB distribution
are essentially integrated with other mitotic events, such as bud emergence and DNA
replication (Byers & Goetsch, 1974, 1975 a). Similarly, the genetic analysis of meiosis
demonstrates an essential integration with SPB behaviour here as well (Moens,
Esposito & Esposito, 1974). The pivotal role of the yeast spindle pole bodies throughout the life cycle therefore provides a particularly useful system among eukaryotes for
the analysis of such organizing centres.
The mechanism initiating microtubule assembly
In the present study, the initiation of microtubule assembly both in vivo and in
vitro results in a structural modification at the end of the microtubule where initiation
has occurred. The differentiation of this closed end from the open appearance distally
might, of course, result from effects other than the initiating mechanism. One might
conceive that the embedment of this end of the microtubule into the material of the
SPB secondarily causes the closed appearance by inhibiting continued polymerization
or depolymerization at this site or by altering its reaction to negative staining. Nevertheless, having found that the proximal end still differs from the distal end after disruption from the SPB, it is not unreasonable to propose that the closed end represents
a structural differentiation persisting at the site of the initiation process after having
served as a 'seed' for tubulin polymerization. By this hypothesis, a cap-like structure,
which will come to comprise the closed end, provides a framework for tubulin polymerization adjacent to its circular rim. This initial ring of tubulin subunits would
thereby favour the contiguous assembly of the typical cylindrical polymer.
The analogous seeding of polymerization by intact singlet (Borisy, Olmsted,
Marcum & Allen, 1974) and doublet (Allen & Borisy, 1974; Burns & Starling, 1974)
microtubules has been shown to accelerate the rate at which elongated microtubules
appear in partially purified neurotubulin extracts. More recently, protein factors
potentiating the formation of nucleation structures for microtubule assembly in more
highly purified neurotubulin extracts have been described (Weingarten, Lockwood,
Hwo & Kirschner, 1975; Murphy & Borisy, 1975; Bloodgood & Rosenbaum, 1976).
Whether or not the ring-shaped polymers of tubulin actually serve as seeds for the
initiation of microtubule assembly as proposed by Borisy & Olmsted (1972) remains
open to question (Kirschner & Williams, 1974). One may also question, in this regard,
whether the neurotubulin system alone is an appropriate model for this initiation
23
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B. Byers, K. Shriver and L. Goetsch
process in non-neural cells. The unusually great distance of axonal microtubules from
the cell body might obviate an initiating mechanism directly associated with the centrioles or with other components of the cell centre but might instead require a unique
mechanism for initiation of polymerization in distal segments of the neuron.
In the present case, the initiation occurring in association with the spindle pole
bodies clearly results in a differentiation of microtubule ends undetected in repolymerized neurotubules alone. Although the closed end is also found in the in vitro
system, the source of its components remains obscure. If it represents a component
of the SPB persisting after the depolymerization of native microtubules, then it must
resist dissociation in sucrose gradients but occasionally be dissociable during spreading
and negative staining. Alternatively, it may consist of tubulin or another disassembled
(slowly sedimenting) component of the neurotubulin extract which is induced by the
SPB to assemble in this manner. If composed of neurotubulin itself, the closed end
would represent a novel tubulin polymer, which has not been detected in the brain
extract alone. Such heteromorphic polymerization of tubulin would not be unprecedented in the assembly of surface lattices; similar contours have been noted in the
closed ends of cylindrical ribosomal polymers (Byers, 1967) as well as in the icosohedral
symmetry at the closed ends of tubular variants of polyoma virus (Kiselev & Klug,
1969).
Regardless of its source, the closed end results from the SPB-associated initiation
of microtubule assembly both in vivo and in vitro. The fact that this initiation is
inhibited by proteolysis suggests that there are essential proteinaceous components.
These might comprise the closed end itself, if it persists on the SPB, or might be
components of a mechanism which secondarily induces formation of this end at the
surface of the SPB. Further analysis in vitro will be required to elucidate the macromolecular mechanisms for the initiation of microtubule polymerization and the
possible generality of these mechanisms to other eukaryotes.
We gratefully acknowledge the financial support of this research by the National Institutes
of Health (GM 18541) and the American Cancer Society (VC-145). K. S. is a predoctoral fellow
supported by the National Institutes of Health.
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{Received 5 January 1977 - Revised 22 July 1977)