Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at 0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.     

Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations.

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborat centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter neculeus reforms in the bud and the other is reformed in the mother cell. Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.     

Functions of microtubules in the Saccharomyces cerevisiae cell cycle

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.     

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.     

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.     

Spc110p: assembly properties and role in the connection of nuclear microtubules to the yeast spindle pole body.

Spc110p is an essential component of the budding yeast spindle pole body (SPB). It binds calmodulin and contains a long central coiled-coil rod which acts as a spacer element between the central plaque of the SPB and the ends of the nuclear or spindle microtubules. This suggests that the essential function of Spc110p is to connect the nuclear microtubules to the SPB. To confirm this, we examined the phenotype of ts alleles of SPC110, one of which contains a mutation in the calmodulin binding site and was suppressed by overexpression of calmodulin. The alleles fail to form a functional mitotic spindle because spindle microtubules are not properly connected to the SPB. We also examined the phenotype of the toxic overexpression of either the wild-type or a truncated version of Spc110p containing a deletion of most of the coiled-coil domain. Both of these proteins form large ordered spheroidal polymers in the nucleus. The polymerization of the truncated Spc110p appears to be initiated inside the SPB from the position where Spc110p is normally located, and as the polymer grows in size it severs the connection between the nuclear microtubules and the SPB. The polymers were purified and are composed of Spc110p and calmodulin. A model for the structure of the polymer is proposed.     

The Spindle Midzone Microtubule-Associated Proteins Ase1p and Cin8p Affect the Number and Orientation of Astral Microtubules in Saccharomyces cerevisiae

The nucleus of the budding yeast S. cerevisiae has to move to the bud neck during mitosis in order for proper DNA segregation to take place. This movement is mediated by spindle and astral microtubules, and it relies on forces generated by microtubule-associated motor proteins. When budding yeast cells express the non-cleavable cohesin subunit, Scc1-RRDD, sister chromatid separation is blocked, preventing the spindle from elongating. Thus, in the presence of Scc1-RRDD nuclear positioning is mediated solely by forces acting through astral microtubules. We have previously shown that under these conditions cells exit mitosis with the nucleus in the mother cells, and that the position of the nucleus is determined, at least in part, by the FEAR pathway, which regulates various aspects of mitotic exit. When the FEAR pathway is inactivated, cells expressing Scc1-RRDD exit mitosis with the nucleus in the daughter cells (referred to as a “daughterly phenotype”). In order to find additional proteins that participate in nuclear positioning, we screened a series of mutant strains for those that displayed a daughterly phenotype when Scc1-RRDD was expressed. The most prominent defects were seen in ase1Δ and cin8Δ mutant cells. Both Ase1p and Cin8p were previously shown to be nuclear and to be involved in spindle function. We show here that deletion of ASE1 or CIN8 causes a defect in SPB separation and leads to an abnormal number of astral microtubules and a change in their orientation within the cell. Taken together, these results suggest that in budding yeast Ase1p and Cin8p affect nuclear positioning through astral microtubule-dependent mechanisms.     

Nuclear export receptor Xpo1/Crm1 is physically and functionally linked to the spindle pole body in budding yeast

The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the gamma-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.     

Reorientation of mispositioned spindles in short astral microtubule mutant spc72Delta is dependent on spindle pole body outer plaque and Kar3 motor protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.     

A novel mechanism of nuclear envelope break-down in a fungus: nuclear migration strips off the envelope

In animals, the nuclear envelope disassembles in mitosis, while budding and fission yeast form an intranuclear spindle. Ultrastructural data indicate that basidiomycetes, such as the pathogen Ustilago maydis, undergo an 'open mitosis'. Here we describe the mechanism of nuclear envelope break-down in U. maydis. In interphase, the nucleus resides in the mother cell and the spindle pole body is inactive. Prior to mitosis, it becomes activated and nucleates microtubules that reach into the daughter cell. Dynein appears at microtubule tips and exerts force on the spindle pole body, which leads to the formation of a long nuclear extension that reaches into the bud. Chromosomes migrate through this extension and together with the spindle pole bodies leave the old envelope, which remains in the mother cell until late telophase. Inhibition of nuclear migration or deletion of a Tem1p-like GTPase leads to a 'closed' mitosis, indicating that spindle pole bodies have to reach into the bud where MEN signalling participates in envelope removal. Our data indicate that dynein-mediated premitotic nuclear migration is essential for envelope removal in U. maydis.     

Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.     

Hrs1p/Mcp6p on the meiotic SPB organizes astral microtubule arrays for oscillatory nuclear movement

Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of the nucleus and cytoplasmic organelles in a cell. In fission yeast, oscillatory movement of the nucleus termed horsetail nuclear movement (HNM) has been observed during meiotic prophase. HNM is led by an astral microtubule array emanating from the spindle pole body (SPB), a centrosome-equivalent organelle in yeasts, aided by the dynein-dynactin complex, and is proposed to facilitate the alignment of homologous chromosomes necessary for efficient meiotic recombination. Here we show that a meiosis-specific SPB component Hrs1p (also known as Mcp6p) is a key molecule to remodel microtubules into the horsetail-astral array (HAA). Deletion of Hrs1p impaired HAA formation, leading to compromised HNM. Ectopic expression of Hrs1p during the mitotic cell cycle resulted in the formation of a HAA-like astral microtubule array, which drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the gamma-tubulin ring complex (gamma-TuRC) as well as with a meiotic SPB component. We propose that Hrs1p facilitates formation of the HAA, responsible for the vigorous HNM, by stabilizing connection between the SPB and minus ends of microtubules.     

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.     

Ultrastructural Features of Mitosis in Anisonema sp. (Euglenida)1

The fine structure of stages in mitosis in a colorless euglenoid, Anisonema sp., reveals that chromosomes remain condensed throughout the life cycle and are attached to the nuclear envelope at interphase. The onset of mitosis is marked by the anterior migration of the nucleus towards the base of the reservoir and by elongation of the nucleolus. The nuclear envelope persists throughout mitosis. Microtubules are generated in the peripheral nucleoplasm adjacent to the envelope and attach to the chromosomes while they are still associated with the envelope. The region of microtubular contact develops into a distinct layered kinetochore as the developing spindle with attached chromosomes separates from the nuclear envelope and moves into the nucleoplasm. The mature spindle consists of a number of subspindles each containing about 8–10 microtubules and a few associated chromosomes. Both chromosomal and non-chromosomal microtubules are present in each subspindle and extend towards the envelope terminating at or near the nuclear pores. Chromosomal segregation is concomitant with nuclear elongation. By late division, an interzonal spindle develops in the dumbbell-shaped nucleus and nucleolar separation occurs. Continued invagination of the nuclear envelope in the region of the interzonal spindle eventually separates the daughter nuclei. A remnant of the interzonal spindle persists in the cytoplasm until cytokinesis.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.     

Components of the yeast spindle and spindle pole body

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.     

Life cycles of yeast spindle pole bodies: getting microtubules into a closed nucleus.

The spindle pole body (SPB) is the principal microtubule organizing center of budding and fission yeast. We have examined SPBs and their associated microtubules from both organisms, using electron microscopy and three-dimensional reconstruction techniques, to identify the structural changes that accompany progression through the cell cycle. In this report, we compare these changes in the two kinds of yeasts and present a model for how microtubules get into a closed nucleus.