THE ROLE OF SPINDLE POLE BODIES AND MODIFIED...

J. Cell Sci. 30, 331-352 (1978) 3 3 !Printed in Great Britain © Company of Biologists Limited igjS

THE ROLE OF SPINDLE POLE BODIES AND

MODIFIED MICROTUBULE ENDS IN THE

INITIATION OF MICROTUBULE ASSEMBLY

IN SACCHAROMYCES CEREVISIAE

BRECK BYERS, KATHLEEN SHRIVERAND LORETTA GOETSCHDepartments of Genetics and Biochemistry,University of Washington, Seattle, Washington, U.S.A.

SUMMARYThe 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 topotentiate the polymerization of microtubules in vitro.

Free SPBs were completely deprived of any detectable native microtubules by incubationin the absence of added tubulin and were then challenged with chick neurotubulin, which hadbeen rendered partially defective in self-initiation of repolymerization. Electron microscopyrevealed 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 numbersafter the first stage of the reaction, it is apparent that there are a limited number of sites for in-itiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysisof protein but not of DNA. The microtubule end proximal to the site of initiation on the SPBis distinguished by a ' closed' appearance because of a terminal component which is continuouswith the microtubule wall, whereas the distal end has the 'open' appearance characteristic offreely repolymerized neurotubules. SPBs which were partially purified on sucrose gradientsretained their ability to initiate the assembly of microtubules with the same structural dif-ferentiation of their ends. The occurrence of closed proximal ends on native yeast microtubulessuggests 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 playan essential role in mitotic and meiotic division as well as in other changes of cellularform (Porter, 1966). In order to understand the fundamental mechanisms responsiblefor nuclear division, it is therefore reasonable to examine the manner by which thefunctional arrangements of microtubules are determined. Morphological evidence hassuggested that microtubules arise at intracellular sites, which are believed to nucleatethe polymerization of the component protein precursor, tubulin (Tilney & Goddard,1970). The behaviour of such sites, termed 'microtubule-organizing centres' byPickett-Heaps (1969), may therefore be pivotal to the control of microtubule-mediatedcellular processes.

Address correspondence to: Dr Breck Byers, Department of Genetics, SK-50, Universityof Washington, Seattle, Washington 98195, U.S.A.

332 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 centresin vitro. Rebhun, Lefebvre & Rosenbaum (1973); Inoue, Borisy & Kiehart, (1974);and Weisenberg & Rosenfeld (1975) have subsequently been able to demonstrate theincorporation of exogenous neurotubulin into the organized structure of mitoticand meiotic spindles. Moreover, it has been clearly demonstrated (Weisenberg, 1974;Snyder & Mclntosh, 1975) that the augmented spindle includes microtubules whichhave arisen entirely de novo at the spindle poles in the in vitro system. Further analysisof the initiation process is hampered, however, by the fact that most initiating siteswithin these spindles are indistinct and are diffusely arranged.

In certain eukaryotes, on the other hand, all of the microtubules radiate from pre-cisely defined regions of densely staining amorphous material. This arrangement isparticularly evident in Saccharomyces cerevisiae (Robinow & Marak, 1966; Moens &Rapport, 1971; Peterson & Ris, 1976), in which all of the microtubules end within thediscrete 'spindle plaques'. These diskoidal bodies, which are embedded in the nuclearenvelope throughout the life cycle, represent a type of spindle pole body (SPB) - theorganelle serving as the focus for microtubule arrangements in a wide range of lowereukaryotes (see review by Kubai, 1975). Fundamental changes in the distribution ofmicrotubules, such as in the formation of the mitotic spindle, are necessarilyfocused upon observable alterations of the SPBs to which they are attached (Byers &Goetsch, 1975 a). In order to understand the mechanisms controlling microtubuledistribution, we have therefore undertaken an investigation of the manner in whichthe SPBs mediate microtubule assembly. The present study has revealed that thedemonstrable initiation of neurotubulin polymerization in association with SPBsin vitro results in a terminal modification of microtubule structure similar to that foundon 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. Meioticstages were obtained with strain 212-1 (Simchen, 1974), which is homozygous for the tem-perature-sensitive allele cdc 4-1 (Hartwell, 1971) and may be arrested in meiosis at the doubleSPB stage by elevation of the temperature to 33-5 °C (Byers & Goetsch, 19756). Vegetativegrowth was maintained in YEPD medium consisting of 1 % Bacto-yeast extract, 2 % Bacto-peptone, and 2% dextrose. Presporulation medium PSP2 (Roth & Halvorson, 1969) consistsof 0-67 % yeast nitrogen base, 1 % yeast extract, 1 % potassium phthalate, and 1 % potassiumacetate supplemented with 40 fig/ml each of adenine and uracil. Sporulation medium SPMconsists 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 wereinnoculated into presporulation medium PSP2 and maintained in logarithmic growth for 2or more days before meiosis was induced by washing the cells twice with sterile water andresuspending them in SPM at 22 °C (Roth & Halvorson, 1969). Electron microscopy of fixedand embedded cells (Byers & Goetsch, 19756) demonstrated that the majority of cells wereat 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 con-sisting 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 for10 min at 22 CC. The cells were then transferred to the spheroplasting medium, consisting of1 M NaCl, 0012 M sodium phosphate-citrate buffer, pH 5-8, and 10% (v/v) glusulase (EndoLaboratories, Inc., Los Angles, California). After incubation for 30 min at 22 °C, the sphero-plasts 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 thespheroplasts was accomplished by resuspending the washed pellet in microtubule polymeri-zation buffer, MPB (see below). This also resulted in lysis of the nuclei, freeing the spindlesor spindle poles in the lysate.

Chick neurotubulin extracts

Neurotubulin was prepared from the brains of 20-day chick embryos by the thermalcycling method of Weisenberg (1972), as modified by the addition of glycerol (Shelanski,Gaskin & Cantor, 1973). The brains were Dounce homogenized in microtubule polymerizationbuffer (MPB) consisting of o-i M 2(i\T-morpholino)ethane sulphonic acid (MES), pH 6-4, 1 mMethyleneglycol-ta's(/?-aminoethylether^ATAT'-tetraacetic acid (EGTA),0-5 mM MgCU and 2 mMguanosine triphosphate (GTP). The homogenate was chilled to 4 °C and centrifuged at 130000 gfor 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 for60 min at 25 °C. Tubulin pellets were quickly frozen by immersion in liquid nitrogen and werestored at —70 °C. Tubulin was resuspended at 5 mg/ml from thawed pellets by Douncehomogenization in one-fourth the original volume of MPB at 4 °C and centrifuged at 4 °Cfor 90 min at 230000 g to yield a 'high-speed' supernatant deficient in the potential for self-initiation 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 minat 4 °C. One-fourth volume of 100% glycerol was added to the tubulin supernatants beforepolymerization tests.

Polymerization of neurotubulin on spindle pole bodies

The assembly of microtubules in association with free SPBs was assayed by either of 2procedures. 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 microtubulesin association with the SPBs adherent to the Formvar film. In procedure B, the lysate was directlymixed with the neurotubulin solution; this mixture was incubated at 20 or 37 °C and sub-sequently sampled by touching it with Formvar-coated grids. In either procedure, the sampleswere then rinsed twice with distilled water and negatively stained by touching the grid to adrop of 1 % uranyl acetate and blotting it dry. Partial disruption of the microtubules from theirassociation with the SPBs was sometimes enhanced by soaking the sample in the negativestain for 5 min before blotting dry. This disruption facilitated observation of the microtubuleends which had been attached to the SPB and had therefore been difficult to observe in the thickerstain immediately surrounding the SPB.

Enzymic hydrolysis

The sensitivity of the initiation potential to enzymic hydrolysis was assayed by a modificationof procedure B; selected enzymes were added to the yeast lysate in MPB and the mixture waspreincubated 30 min at 30 °C before addition of neurotubulin. Deoxyribonuclease I and ribo-nuclease A (Worthington) were used at final concentrations of 50 /*g/ml and 1 o/tg/ml, respectively.The efficacy of these nuclease treatments was determined by electron microscopy (see Results).

22 CEL 30


Trypsin (Sigma, type V) was used at 10 /tg/ml and its activity was arrested at the end of thepreincubation by the addition of 20 /tg/ml soybean trypsin inhibitor (Sigma, type II-S). Theproteolytic activity of the trypsin in MPB was assayed by the spectrophotometric analysis ofa-JV-benzoyl-AT-arginine ethyl ester HC1 (Sigma). Under these buffer conditions, the trypsintreatment represented 15 BAEE units of activity (14% of the standard activity at pH 7-6) andwas 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 at37 °C in 0-05 mg/ml deoxyribonuclease I before being layered on to a continuous gradient of0-3 to i-8 M sucrose in MPB. Following centrifugation at 30000 rev/min at 4 °C for 30 minin a Spinco SW41 rotor, the gradients were fractionated by pumping the solution from thebottom. Aliquots of the fractions were analysed for their optical density at 230 nm to detectcomponents of the lysate and by optical refraction to characterize the gradient of sucrosedensity. Other aliquots were tested for their content of SPBs competent to induce microtubulepolymerization by applying a droplet to a grid and inverting the grid on a droplet of high-speed supernatant tubulin for a 10-min incubation at 37 °C. The grids were then negativelystained with 1 % uranyl acetate and the number of microtubule-associated SPBs per 10 gridsquares was determined by electron microscopy.

RESULTS

Electron microscopy of lysates

Spindle pole bodies (SPBs) were easily identified in negatively-stained lysates ofspheroplasts prepared from vegetative yeast cultures, confirming earlier observationsof Borisy, Peterson, Hyams & Ris (1975) and Peterson & Ris (1976). If spheroplastswere lysed directly on the water surface to which electron-microscope grids were thenapplied, the SPBs were almost invariably found in the vicinity of the released chrom-atin. Microtubules were found to remain attached to the SPBs in the same configur-ation as that seen in intact cells by thin-section electron microscopy. Lysates ofactively growing vegetative cultures included identifiable single SPBs, double SPBs(which had duplicated but not undergone separation), and complete spindles about1 /tm long. Longer spindles, known from thin-section electron microscopy to ariselate in nuclear division, were rarely seen; this stage therefore appears to undergoselective loss during spheroplasting (Peterson & Ris, 1976).

Lysates of spheroplasts prepared from early meiotic stages gave a better yield ofinterpretable preparations than those from vegetative cells because the chromatinwas less adherent and rarely obscured the microtubules. Stages similar to those inthe 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 meioticcells (strain 212-1 at double SPB stage after 6 h in meiosis). A, negative staining im-mediately after lysis reveals microtubules with closed ends (ce) proximal to the doubleSPB (one arrow to each element) and open ends (pe) located distally. B, greater dis-ruption of microtubule-SPB attachment reveals several closed ends (ce) adjacent to theSPB. C, microtubule-free double SPB after 15 min incubation in MPB before staining,x 65000.


pb

336

2A

B. Byers, K. Shriver and L. Goetsch

B

Fig. 2. Pairs of SPBs freed by lysis of cells in meiosis I (at 8 h in meiosis). A, SPBsfreed from a single spindle by incubation in MPB to remove native microtubules.x 80000. B, microtubule addition to same preparation after incubation with neuro-tubulin. x 15000. spb, spindle pole body; v, vesicles.


1971). The SPBs measure about 50 nm in the dimension parallel with the attachedmicrotubules and 300 nm in the dimension perpendicular to the microtubules. Theseand other structural features are consistent with the morphology of these bodies asseen in thin sections. In addition, however, the negatively-stained preparations re-vealed a previously undetected modification of mierotubule structure. This modi-fication was revealed by the difference in the distribution of the negative stain at the2 ends of each mierotubule (Fig. 1 A, B). The end distal to the SPB has the appearancetypical of microtubules prepared from neurotubulin alone, the stain being uniformlydistributed in the axial region and occasionally revealing the frayed protofilaments.This distribution of the stain indicates that the distal mierotubule end may be definedas the end of an open cylinder, there being no interruption at the end of the miero-tubule between the lumen and the exterior. We will henceforth designate ends of thisappearance as'open'. The end attached to the SPB, on the other hand, appears to lackany opening to the exterior but instead consists of a closed surface which is continuouswith the mierotubule wall and excludes the stain to the same extent. We will designatethis end as 'closed'. By analogy, the yeast spindle mierotubule could be likened to arimless test tube, the open top of the test tube simulating the open end distal to theSPB and its closed bottom simulating the closed proximal end of the mierotubule. Theclosed end of the mierotubule sometimes displays the even curvature seen in the testtube bottom, but is often more angular in profile (as may be noted in some views inFig. IB).

The polarity of these microtubules with respect to their SPB attachment was derivedfrom 2 sorts of observations. In the first place, observations of intact spindle polesshowed that the proximal ends were usually embedded so deeply in the negative stainsurrounding the SPB that their form could not be seen. Those few which could beseen were all of the closed form, whereas the distal ends were easily seen and wereinvariably open. Secondly, the occasional partial disruption of spindle poles permittedunambiguous observation of the proximal ends of the microtubules. When the at-tachment between the proximal end and the SPB was disrupted, the proximal endwas 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 derivedby breakage because they had 2 open ends, one of which was adjacent to the open endof another mierotubule. The remaining majority of well stained microtubules wereseen to have one closed end, which was almost always nearer the SPB than was theopen end. The minor fraction (less than 5%) of the opposite orientation must havebeen inverted upon disruption because the distal ends of attached microtubuleswere never (in 220 unambiguous observations) of the closed form. These observationstherefore indicate that the proximal end bears a distinct structural modification.Whether this modification is associated with attachment to the SPB or with the initia-tion of polymerization at this site will be considered later.


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 incell-free lysates. Upon examining the depolymerization of native yeast microtubules,we found that lysis of cells in microtubule buffer (Weisenberg, 1972) followed byincubation at 37 °C for 5 min or longer resulted in the complete loss of any micro-tubules detectable by electron microscopy (Figs. 1 c, 2A). This loss made the SPBsdifficult to detect with certainty, but certain structural features permitted theiridentification. Bodies of the appropriate form and dimensions were frequently seenin the vicinity of the chromatin, where microtubule-laden SPBs would be found inunincubated samples. Moreover, a single SPB could often be identified by the charac-teristic 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 withcircular vesicles, 28-30 nm in diameter, which were clearly revealed by negativestaining. This association is discussed more fully later.

The ability of these microtubule-free SPBs to initiate the polymerization of tubulinwas then tested. Lacking a system of yeast tubulin competent to repolymerize invitro, we used chick neurotubulin with a high potential for repolymerization. Afterone cycle of polymerization in a low-speed supernatant, the microtubules weredepolymerized in the cold and the tubulin was subjected to more extensive ultra-centrifugation in order to reduce its potential for self-initiation of polymerization(Borisy & Olmsted, 1972). This tubulin was then combined with lysates containingmicrotubule-free spindle pole bodies either after adhesion of the SPBs to Formvarfilms on electron-microscope grids (procedure A) or in free suspension (procedure B). Ineither case, electron microscopy demonstrated that incubation at 37 °C resulted in thepolymerization of microtubules in association with the SPBs (Figs. 2B, 3).

Initial experiments on total yeast lysates were difficult to interpret because themicrotubules were often entangled in the masses of chromatin adjacent to the SPBs.It was unclear whether this entanglement represented (1) some initiation of poly-merization within the chromatin (perhaps at kinetochores), (2) the specific adhesionof microtubules to chromatin, or (3) non-specific tangling. In any case, the confusionof chromatin-associated microtubules was eliminated in subsequent experiments byadding deoxyribonuclease to the preincubation mixture used to depolymerize nativemicrotubules. This treatment removed most visible chromatin from the Formvarfilms but did not hinder the subsequent initiation of microtubule polymerization onthe SPBs. In these preparations, the vast majority of microtubules could easily be seento arise from the SPBs. The number of microtubules in the background (unassociatedwith the SPBs) varied with the tubulin preparation but was usually less than 10 pergrid square (2500/«m2). In a typical preparation, the material released from two tofour spheroplasts was seen within one grid square. Each area of lysis contained oneduplicated SPB with 20-30 microtubules radiating from each half of the double SPB.

Yeast spindle pole bodies

B

339

Fig. 3. Time course of neurotubulin polymerization in vitro on to SPBs (after de-polymerization 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.

34° B. Byers, K. Shriver and L. Goetsch

Therefore, over 90% of the repolymerized microtubules seen were associated withthe identifiable SPBs.

Control experiments established that microtubule formation in this mixed systemwas subject to constraints similar to those of a simple neurotubulin repolymerizationsystem. No microtubules appeared in normal preparations maintained at o °C, nordid microtubules form if o-i mM colchicine was added to the preparation before in-cubation at 37 °C. Therefore, the in vitro system has the same sensitivity to cold andcolchicine as does the neurotubulin system alone, although yeast microtubules persistat this concentration of colchicine in vivo (Haber, Peloquin, Halvorson & Borisy,1972).

40Time, min

Fig. 4. Microtubule number (upper panel) and microtubule length (lower panel) inthe time course of neurotubulin incubation in vitro with mixed single and doublespindle pole bodies (from lysed spheroplasts of strain 212-1 after 6 h in meiosis)preincubated in MPB to remove native microtubules. Each time point representsmeasurements on 20 SPBs, and vertical bars indicate the limits of one standarddeviation for these data.

Properties of microtubule repolymerization

In order to determine the role of the spindle pole bodies in the reformation of micro-tubules 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 super-natant neurotubulin and incubated at 37 °C. Samples were taken by touching gridsto the mixture at successive times of incubation; the grids were then stained for


electron microscopy. This time course ofrepolymerization demonstrated that initiationwas 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 notremnants of the native spindle but are newly initiated in vitro. Furthermore, the over-all length of the microtubules eventually formed (Fig. 4) far exceeds the length ofthe native microtubules originally present in the yeast cell. It is therefore obviousthat at least the majority of each microtubule was formed in vitro.

The same samples were also used to determine the increase in microtubule numbersemanating from each SPB during the incubation. Because unfavourable orientationor staining often prevented our determining whether or not an individual SPB hadundergone duplication (in preparation for meiosis I), we simply counted the micro-tubules 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 signi-ficantly after the first 5-10 min of incubation, but persisted at a stable value as themicrotubules continued to elongate. One must conclude, therefore, that the potentialfor initiation of microtubule assembly had become saturated in spite of adequateneurotubulin for extensive further elongation. These data also show that it is unlikelythat microtubules are simply aggregating on the SPBs rather than being initiatedthere. If aggregation were occurring, it would have had to undergo completion withinthe first few minutes of incubation.

Macromolecular nature of the initiating sites

After finding that spindle pole bodies devoid of visible microtubules could initiateneurotubulin polymerization, we attempted to determine what macromolecular com-ponents of the SPBs were essential to the initiation process. We had already found thatdeoxyribonuclease treatments sufficient to disrupt all visible chromatin did not preventinitiation of polymerization. Similar tests with ribonuclease A were complicated bythe fact that this enzyme may have a stimulatory effect on neurotubulin repoly-merization (Bryan, Nagle & Doenges, 1975). The treatments employed (see Methods),which were sufficient to eliminate all visible ribosomes from electron-microscopicpreparations of the lysate, appeared to stimulate the formation of microtubules inassociation with the SPBs, but the number of unattached (background) microtubuleswas also increased to such an extent that the apparent effect of ribonuclease on SPB-associated polymerization remained inconclusive.

The requirement for trypsin-sensitive components was, on the other hand, clearlydemonstrable. Initial tests with 15 BAEE trypsin units per ml during preincubationand incubation with tubulin resulted in a failure to form microtubules, probablybecause of the direct effect of trypsin on the tubulin itself. This effect was sub-sequently avoided by adding soybean trypsin inhibitor after preincubation in trypsin,but before the addition of tubulin. Under these conditions, the usual numbers of freemicrotubules were seen to have arisen in the background but none was found attachedto the SPBs, thereby demonstrating the trypsin-sensitivity of their potential for in-itiation. The absence of any inhibitory effect by trypsin and trypsin inhibitor togetherwas proven by the substantial initiation of microtubules if both trypsin and inhibitor


Fig. S- Differentiation of ends of microtubules assembled in vitro (as in Fig. 3) for 15min. A, prolonged uranyl acetate negative stain has dissociated several microtubulesfrom the SPB. x 24000. B, detailed view of proximal closed ends similar to those ofnative microtubules. x 70000. c, detailed view of distal open ends, x 70000. ce,closed end; oe, open end; v, vesicles.


were present throughout both the preincubation and the subsequent incubation withneurotubulin. We conclude that the potential to initiate polymerization is sensitive totrypsin and insensitive to deoxyribonuclease as well as, perhaps, to ribonuclease.These results suggest that sites responsible for the initiation of polymerization de-pend upon the integrity of proteinaceous components.

Anatomy of the repolymerized microtubules

The microtubules induced to assemble in vitro were similar in appearance to thenative yeast microtubules originally seen in simple lysates. Even when the lysate waspreincubated in the absence of neurotubulin, so that no remnants of native micro-tubules were detectable by electron microscopy, the microtubules which formedupon subsequent addition of neurotubulin were closed at the proximal end. Althoughthe microtubule ends attached to the SPB were rarely detectable in the dense negativestain in this region, all proximal ends seen were of the closed form. As in the case ofnative microtubules, the proximal ends were more clearly seen if they had becomedetached from the SPB during negative staining (Fig. 5). Detached microtubuleswere found to have only one closed end, which usually remained nearer the detachedSPB. It is probable that the occasional microtubule with its closed end distal to theSPB had become inverted from its original orientation, because the distal ends of thereassembled microtubules which had remained attached were invariably of the openform. These distal ends were frequently also splayed, revealing separated protofilamentsor 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 spheroplastsremain as discrete bodies which will, when challenged with neurotubulin competentto repolymerize, stimulate the assembly of microtubules. In order to establish whetherthis stimulation of assembly depends upon functions residing within the SPBs alone,gradient separation of these bodies from soluble components of the lysate was under-taken.

Spheroplasts were lysed by dilution in MPB with no added sorbitol and were layeredon to sucrose gradients. Initial attempts at the centrifugal separation of lysate com-ponents were hindered by the fact that the DNA freed from lysed nuclei made a viscouslayer which trapped other components. Because the potential for initiation of micro-tubule assembly was insensitive to deoxyribonuclease, subsequent samples were treatedwith this enzyme as described. Following centrifugation, fractions of the gradientswere touched to grids, which were in turn applied to high-speed supernatant tubulinin order to induce the assembly of microtubules. Negative staining and electron micro-scopy then revealed the distribution of SPBs among the fractions.

In earlier experiments on simple lysates, a centre from which microtubules radiatedcould be identified as an SPB not only by its anatomy but because of its presencewithin the localized debris released by lysis of a single spheroplast. Because this debriswas dispersed and partially removed during the gradient procedure, the identification


ce

i,

Fig. 6. Microtubules polymerized in association with gradient-separated SPBfractions. A, pairs of SPBs (double arrows) released from strain 501 iD cells after 3 h ofarrest 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.


of centres of radiating microtubules as SPBs relied solely on their anatomy. We didfind, in fact, that we could often identify the 2 components of the duplicated SPB ifthe gradient had been prepared from spheroplasts of meiotic cells at this stage, butunfavourable staining or angle of view frequently obscured observations to thisresolution. On the other hand, this ambiguity was not encountered in the analysis ofgradients of lysates of strain 5011D (temperature-sensitive for cdc 24) after temperaturearrest of vegetative growth. Spheroplasts of these cells regularly release large numbersof intact complete spindles; their microtubules rapidly undergo depolymerization but

10 15Fraction

Fig. 7. Isopycnic sucrose gradient separation of double SPB fraction from other com-ponents of lysed spheroplasts prepared after 6 h in meiosis (see Methods). The bottomof the gradient is at the left. Application of droplets of fractions to grids followed bychallenge with neurotubulin reveals a maximum concentration of free SPBs (closedcircles in upper panel) in fraction 6 (146 M sucrose), whereas the bulk of debris indi-cated by light scattering (O.D.230 nm, open circles) is maximal in fraction 7 (1-40 Msucrose). Low molecular weight components remain in fractions 15-20 at the top of thegradient. A> sucrose concentration.

their SPBs remain attached to one another in pairs, apparently interconnected byremnants of the nuclear envelope or nuclear cortex. Challenge of these lysates withtubulin results in the appearance of distinct pairs of centres from which microtubulesradiate (Fig. 6A). The 2 members of each pair lie less than 1-5 /«m apart (more than100 observations) whereas each pair is usually separated from the next by more than8 /mi. This decidely non-random distribution of the centres is consistent with theexpected pairing of SPBs, further establishing the identity of these centres of assemblywith the SPBs themselves.

Whether from meiotic or vegetative cultures, the SPBs were consistently found to


form a band at an apparent density slightly greater than the peak of light-scatteringmaterial (Fig. 7). These partially purified SPBs retained the ability to induce micro-tubule assembly as rapidly as those recovered directly from unseparated lysates.Moreover, these microtubules again bore a closed end adjacent to the site of initiationat the SPB (Fig. 8). Although the current procedures do not yet permit a more thoroughpurification, this partial separation from other components - particularly from the lowmolecular weight components remaining at the top of the gradient — indicates that theinduction of microtubule assembly by a mechanism producing a closed proximal endresides principally in the spindle pole bodies alone.

Vesicles associated with spindle pole bodies

SPBs freed from lysed spheroplasts were usually found associated with distinctcircular vesicles, 28-30 nm in diameter (Fig. 2A). From 0 to 12 of these vesicles werefound near each SPB, but they were only rarely seen at greater distances. They wereoccasionally found adjacent to SPBs which retained their native microtubules andwere often seen in association with those upon which neurotubulin had been poly-merized (Fig. 5B). They were also found in association with SPBs subjected to sucrosegradient centrifugation (Fig. 6 B), demonstrating the persistence of their associationwith the SPBs under these conditions in vitro. The role of these vesicles is not evidentfrom the present observations.

DISCUSSION

Every microtubule in Saccharomyces cerevisiae appears to have at least one of itsends associated with the spindle pole bodies (SPBs) during both mitosis (Robinow &Marak, 1966) and meiosis (Moens & Rapport, 1971). In the present study, we haveinvestigated the mechanisms which may be responsible for the generation of thisarrangement of microtubules. Negative staining of SPBs freed from spheroplasts byosmotic 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 endis found to consist of a closed hemispherical, or sometimes angular, surface continuouswith the microtubule wall. We have designated the former type of ends as open andthe latter as closed.

Incubation of the lysate in microtubule assembly buffer resulted in the disassemblyof the native microtubules. Subsequent challenge of the lysate with avian neurotubulinresulted 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 initiationof 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 frommeiotic cells (as in Fig. 6B). A, attached microtubules have open ends distally (openarrows), x 90000. B-F the closed ends (solid arrows) of these microtubules disruptedfrom SPBs were nearer the SPB than the open ends (open arrows), x 110000.


study, dealing principally with meiotic cells, we have found in addition that the prox-imal ends of microtubules formed in vitro display a closed configuration similar tothat present in native yeast microtubules. Moreover, we have noted that the numberof microtubules arising on each SPB in vitro achieves saturation early in the reactionin spite of continued elongation of the microtubules present. This saturationphenomenon suggests that each SPB may bear a limited number of discrete sites for theinitiation of microtubule assembly. Each such site may then provide for the initiation of asingle microtubule with a closed end adjacent to the SPB, thereby accounting forthe fixed number of native spindle microtubules found by electron microscopy ofwhole cells (Peterson & Ris, 1976).

We have also noted that spherical vesicles, 28-30 nm diameter, are found associatedwith the spindle pole bodies upon microtubule depolymerization. These are similarin appearance to vesicles frequently seen in association with SPBs in thin sections ofwhole cells (unpublished observations). Their association with a site for the initiationof microtubule assembly also suggests some similarity to large vesicles recently notednear the spindle poles of mammalian cells (Gould & Borisy, 1976) but it remainsunknown 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 ofdensely staining amorphous material (Tilney, 1971) similar in appearance to thatcomprising the spindle pole bodies. For example, Brown & Bouck (1973) have demon-strated that the extensive arrays of microtubules in the flagellate Ochromonas terminatein association with dense material of this type. The initiation of microtubule repoly-merization following transient depolymerization in vivo appears to occur at these sites.Stearns, Connolly & Brown (1976) have more recently described similar material inassociation with the basal bodies of the alga Polytomella agilis and have further demon-strated the ability of this material to potentiate the initiation of neurotubulin poly-merization in vitro. These observations suggest that the initiating function serves asthe mechanism controlling microtubule distribution both in vivo and in vitro. Ifmicrotubule distribution is, indeed, determined by initiating sites, then the funda-mental basis for control must be sought in the processes which redistribute andregulate the activity of these sites. One may then envisage that the varied sites con-trolling microtubule polymerization undergo reorganization in the course of the cellcycle. During mitosis in animal cells, for example, some sites are discretely localizedat the kinetochores (Telzer, Moses & Rosenbaum, 1975), whereas many sites mustoccur in the region immediately surrounding the centrioles. During interphase, onthe other hand, the controlling sites must reside in more widely dispersed regions ofthe cell. Direct observations on the behaviour of the 'pericentriolar satellites' duringmitosis of HeLa cells demonstrates, in fact, that these structures, which are clearlyassociated with the ends of some of the microtubules, undergo sequential changes intheir distribution (Robbins, Jentzsch & Micali, 1968). The precise analysis of thisredistribution is hampered, however, by the indistinct morphology and inadequatelydefined staining properties of the satellites.


By contrast, all apparent centres for microtubule organization in S. cerevisiae areclearly defined throughout the entire life cycle. Every microtubule in the cell ends ata discrete spindle pole body. Therefore, any redistribution of microtubules is neces-sarily accompanied by changes in these readily observable sites (Byers & Goetsch,1974). During vegetative growth, for instance, the single SPB duplicates in coincidencewith bud emergence and cytoplasmic microtubules extend from the outer surface ofthe 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 ofthe nucleus until the mitotic spindle forms between the separating SPBs. The terminalphenotypes of several temperature-sensitive cell division cycle mutants isolated byHartwell, Culotti & Reid (1970) demonstrate that specific changes in SPB distributionare essentially integrated with other mitotic events, such as bud emergence and DNAreplication (Byers & Goetsch, 1974, 1975 a). Similarly, the genetic analysis of meiosisdemonstrates an essential integration with SPB behaviour here as well (Moens,Esposito & Esposito, 1974). The pivotal role of the yeast spindle pole bodies through-out the life cycle therefore provides a particularly useful system among eukaryotes forthe analysis of such organizing centres.

The mechanism initiating microtubule assembly

In the present study, the initiation of microtubule assembly both in vivo and invitro results in a structural modification at the end of the microtubule where initiationhas occurred. The differentiation of this closed end from the open appearance distallymight, of course, result from effects other than the initiating mechanism. One mightconceive that the embedment of this end of the microtubule into the material of theSPB secondarily causes the closed appearance by inhibiting continued polymerizationor depolymerization at this site or by altering its reaction to negative staining. Never-theless, having found that the proximal end still differs from the distal end after dis-ruption from the SPB, it is not unreasonable to propose that the closed end representsa structural differentiation persisting at the site of the initiation process after havingserved 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 poly-merization adjacent to its circular rim. This initial ring of tubulin subunits wouldthereby 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 microtubulesappear in partially purified neurotubulin extracts. More recently, protein factorspotentiating the formation of nucleation structures for microtubule assembly in morehighly 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 theinitiation of microtubule assembly as proposed by Borisy & Olmsted (1972) remainsopen to question (Kirschner & Williams, 1974). One may also question, in this regard,whether the neurotubulin system alone is an appropriate model for this initiation

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process in non-neural cells. The unusually great distance of axonal microtubules fromthe cell body might obviate an initiating mechanism directly associated with the cen-trioles or with other components of the cell centre but might instead require a uniquemechanism for initiation of polymerization in distal segments of the neuron.

In the present case, the initiation occurring in association with the spindle polebodies clearly results in a differentiation of microtubule ends undetected in repoly-merized neurotubules alone. Although the closed end is also found in the in vitrosystem, the source of its components remains obscure. If it represents a componentof the SPB persisting after the depolymerization of native microtubules, then it mustresist dissociation in sucrose gradients but occasionally be dissociable during spreadingand negative staining. Alternatively, it may consist of tubulin or another disassembled(slowly sedimenting) component of the neurotubulin extract which is induced by theSPB to assemble in this manner. If composed of neurotubulin itself, the closed endwould represent a novel tubulin polymer, which has not been detected in the brainextract alone. Such heteromorphic polymerization of tubulin would not be unpre-cedented in the assembly of surface lattices; similar contours have been noted in theclosed ends of cylindrical ribosomal polymers (Byers, 1967) as well as in the icosohedralsymmetry 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 initiationof microtubule assembly both in vivo and in vitro. The fact that this initiation isinhibited by proteolysis suggests that there are essential proteinaceous components.These might comprise the closed end itself, if it persists on the SPB, or might becomponents of a mechanism which secondarily induces formation of this end at thesurface of the SPB. Further analysis in vitro will be required to elucidate the macro-molecular mechanisms for the initiation of microtubule polymerization and thepossible generality of these mechanisms to other eukaryotes.

We gratefully acknowledge the financial support of this research by the National Institutesof Health (GM 18541) and the American Cancer Society (VC-145). K. S. is a predoctoral fellowsupported by the National Institutes of Health.

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{Received 5 January 1977 - Revised 22 July 1977)

THE ROLE OF SPINDLE POLE BODIES AND MODIFIED...

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