Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus,Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.     

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.     

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.     

Microtubular and actin cytoskeletons and ultrastructural characteristics of the potentially pathogenic basidiomycetous yeast Malassezia pachydermatis

Microtubular and actin cytoskeletons were investigated in the lipophilic yeast Malassezia pachydermatis by fluorescence and electron microscopy. To detect microtubules by indirect immunofluorescence using monoclonal anti-tubulin antibody, a prolonged incubation with lysing enzymes was necessary due to its very thick cell wall. Cytoplasmic microtubules were detected in interphase and a spindle with astral microtubules was seen in M-phase. The disintegration of cytoplasmic microtubules and migration of the nucleus to the bud before mitosis were characteristic features of the basidiomycetous yeast Malassezia pachydermatis. The visualisation of F-actin structures (patches, cables and cytokinetic rings) by fluorescence microscopy using both monoclonal anti-actin antibody and rhodamine-phalloidin failed, but actin was detected by electron microscopy with immunogold labelling. Clusters of gold particles indicating actin structures were detected at the plasma membrane of cells with unique cortical ultrastructural features characteristic of the genus Malassezia. A possible association of these with the actin cytoskeleton is suggested.     

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.     

Actin-mediated Delivery of Astral Microtubules Instructs Kar9p Asymmetric Loading to the Bud-Ward Spindle Pole

In Saccharomyces cerevisiae, Kar9p, one player in spindle alignment, guides the bud-ward spindle pole by linking astral microtubule plus ends to Myo2p-based transport along actin cables generated by the formins Bni1p and Bnr1p and the polarity determinant Bud6p. Initially, Kar9p labels both poles but progressively singles out the bud-ward pole. Here, we show that this polarization requires cell polarity determinants, actin cables, and microtubules. Indeed, in a bud6Δ bni1Δ mutant or upon direct depolymerization of actin cables Kar9p symmetry increased. Furthermore, symmetry was selectively induced by myo2 alleles, preventing Kar9p binding to the Myo2p cargo domain. Kar9p polarity was rebuilt after transient disruption of microtubules, dependent on cell polarity and actin cables. Symmetry breaking also occurred after transient depolymerization of actin cables, with Kar9p increasing at the spindle pole engaging in repeated cycles of Kar9p-mediated transport. Kar9p returning to the spindle pole on shrinking astral microtubules may contribute toward this bias. Thus, Myo2p transport along actin cables may support a feedback loop by which delivery of astral microtubule plus ends sustains Kar9p polarized recruitment to the bud-ward spindle pole. Our findings also explain the link between Kar9p polarity and the choice setting aside the old spindle pole for daughter-bound fate.     

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.     

Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast

The orientation of the mitotic spindle plays a key role in determining whether a polarized cell will divide symmetrically or asymmetrically. In most cell types, cytoplasmic dynein plays a critical role in spindle orientation. However, how dynein directs opposite spindle poles toward distinct and predetermined cell ends is poorly understood. Here, we show that dynein distributes preferentially to the spindle pole bodies (SPB) and astral microtubules (MTs) proximal to the bud in metaphase yeast cells. Dynein asymmetry depended on the bud neck kinases Elm1, Hsl1, and Gin4, on the spindle pole components Cnm67 and Cdk1, and on the B-type cyclins Clb1 and Clb2. Furthermore, phenotypic and genetic studies both indicated that dynein is unable to orient the spindle when it localizes to both poles and associated microtubules. Together, our data indicate that proper orientation of the spindle requires dynein to act on a single spindle pole.     

Cytoskeletal structures,ultrastructural characteristics and the capsule of the basidiomycetous yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis>

The cytoskeleton, capsule and cell ultrastructure were studied during the cell cycle of Cryptococcus laurentii. In an encapsulated strain, cytoplasmic microtubules and a mitotic spindle were detected. Mitosis was preceded by migration of the nucleus into the bud. F-actin failed to be visualised by rhodamine-phalloidin (RhPh) in encapsulated cells and therefore an acapsular strain was used. The following actin structures were found: actin dots, actin cables and cytokinetic ring. Ultrastructural studies showed the presence of a nucleus in the bud before mitosis. A collar-shaped structure was seen at the base of bud emergence. A lamellar cell wall and a rough outer surface of the cells were detected. Cytoskeletal structures found in C. laurentii are similar to those in Cryptococcus neoformans, which is a serious human pathogen.     

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.     

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.     

Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Delta cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.     

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.     

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.     

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.     

Somatic nuclear division in the sporidia of Ustilago violacea. IV. Microtubules and the spindle-pole body.

In unbudded cells of the anther smut fungus Ustilago violacea there is a dome-shaped spindle-pole body (SPB) consisting of a core 0.1 mum in diameter surrounded by a ribosome-free region 0.3-0.4 mum in diameter lying in a pocket of the nuclear membrane. After budding the nucleus moves towards the bud and begins to rotate rapidly. At about this stage the SPB divides into two parallel bars each about 0.1-0.15 mum in diameter and 0.3 mum long, separated by a distance of about 0.3 mum. Microtubules associated with the nuclear membrane but not with the SPB are present at the time of nuclear rotation. These microtubules disappear when rotation stops. Microtubules attached to the SPB are found during migration of the chromatinic portion of the nucleus into the bud cell. These microtubules disappear when migration stops and the nuclear membrand begins to break down. The twin SPB bars appear to move into the nucleus through a break in the membrane and begin to move apart forming a spindle about 1 mum long. Chromosomal microtubules (one per kinetochore) were found in several serial sections, and in addition there appeared to be several continuous microtubules present. The separation of the two chromatinic masses appeared to result from elongation of the continuous microtubules to about 3 mum long. Cytoplasmic microtubules and spindle microtubules were both found attached to the SPB as it elongated and one nucleus returned to the mother cell. The paper concludes with a discussion of the SPB as a multifuncitonal control center affecting nuclear migration, spindle formation, membrane breakdown and synthesis, karyogamy, conjugation, budding, chromosomal movement, replication, and disjunction.     

Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex.

Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the 'old' SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly.     

Spindle Polarity in S. cerevisiae: MEN Can Tell

Spatial coordination between the axis of the spindle and the division plane is critical in asymmetric cell divisions. In the budding yeast S. cerevisiae, orientation of the mitotic spindle responds to two intertwined programs dictating the position of the spindle poles: one providing the blueprint for built-in pole asymmetry, the other sequentially confining microtubule-cortex interactions to the bud and the bud neck. The first program sets a temporal asymmetry to limit astral microtubules to a single pole prior to spindle pole separation. The second enforces this polarity by allowing these early formed microtubules to undergo capture at the bud cell cortex while stopping newly formed microtubules once cortical capture shifts to the bud neck. The remarkable precision of this integrated program results in an invariant pattern of spindle pole inheritance in which the "old" spindle pole is destined to the bud. An additional layer of asymmetry is superimposed to couple successful chromosomal segregation between the mother and the bud with mitotic exit. This is based on the asymmetric localization to the committed daughter-bound pole of signaling components of the mitotic exit network. This system operates irrespective of intrinsic spindle polarity to ensure that it is always the pole translocating into the bud that carries the signal to regulate mitotic exit.

Key Words:

Cell cycle, Polarity, checkpoint, Microtubule, Cortical cues     

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 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.