During asymmetric cell division, spindle positioning is critical to ensure the unequal segregation of polarity factors and generate daughter cells with different sizes or fates. In budding yeast the boundary between mother and daughter cell resides at the bud neck, where cytokinesis takes place at the end of the cell cycle. Since budding and bud neck formation occur much earlier than bipolar spindle formation, spindle positioning is a finely regulated process. A surveillance device called the SPOC (spindle position checkpoint) oversees this process and delays mitotic exit and cytokinesis until the spindle is properly oriented along the division axis, thus ensuring genome stability.
Establishment of cell polarity during asymmetric cell division
Asymmetric cell division is widespread in Nature and generates unequal daughter cells with different size and/or fate. This modality of cell division is characteristic of several unicellular organisms, either prokaryotic (such as the bacterium Caulobacter crescentus) or eukaryotic (such as the budding yeast Saccharomyces cerevisiae), and is essential for embryonic development of multicellular eukaryotes. A paradigmatic example of asymmetric cell division is that of stem cells, which generate one daughter cell committed to differentiate and another stem cell that maintains its identity and self-renewing potential. This contributes to organ formation during development and to the homoeostasis of tissues during adulthood. Disrupting asymmetric cell division can lead to uncontrolled cell proliferation and, ultimately, to cancer (reviewed in ).
In order to divide asymmetrically, cells localize polarity factors on one side of the cell, position the mitotic spindle along the polarity axis and specify the division plane with respect to these events. In this way, cell fate determinants are segregated during mitosis to only one of the two daughter cells, making them different from each other.
The basic mechanisms that allow polarity establishment are conserved and initiate with the determination of a spatial cue on the cell surface that defines the point in the cell toward which the cell orientates. The axis of polarity is then propagated from this landmark throughout the cell by adaptation of core mechanisms that assemble and orientate the actin cytoskeleton around the landmark, in turn contributing to proper mitotic spindle positioning, also through assembly of polarized protein scaffolds .
Asymmetric cell division has been studied to a great extent in invertebrate model systems, such as Drosophila melanogaster neuroblasts and Caenorhabditis elegans embryos. The budding yeast S. cerevisiae has also proven to be an excellent model system to investigate the molecular mechanisms governing asymmetric cell division. Budding yeast divides asymmetrically, producing daughter cells (originating from the buds) that at the moment of division are smaller than their mothers. It has been proposed that the bud might be the primitive stem cell equivalent in yeast, in that it can divide for a greater number of generations than its mother . The axis of cell polarity is established during the G1-phase of the cell cycle through localization of determinants that define the site of bud emergence to a particuliar site at the cell cortex that depends on the site of the previous cell division. At the incipient bud site, a landmark in the cell cortex is recognized by Rsr1/Bud1, a Ras-like GTPase that conveys the positional information to the polarity establishment machinery (reviewed in ). The active GTP-bound form of Rsr1 promotes Cdc42 (cell division cycle 42) local activation at the chosen site. Cdc42 is a Rho family GTPase involved in cell polarization, controlling actin cytoskeleton organization, septin recruitment to the future bud site and septin ring assembly . Cdc42 plays a central role in establishing and maintaining cell polarity in animal cells also: as a result of Cdc42 activation, PAR proteins, specifically involved in polarity establishment, become asymmetrically localized to one side of the cell in C. elegans, D. melanogaster and mammals. Alterations of PAR proteins impede the formation of a polarity axis inside the cell and cause defects in spindle positioning and orientation in C. elegans and D. melanogaster, indicating that polarity establishment is a crucial step in asymmetric cell division of all eukaryotic cells .
After polarity establishment, the mitotic spindle must be correctly aligned with respect to the polarity axis in order to ensure accurate chromosome segregation. Budding yeast undergoes a closed mitosis, without nuclear envelope breakdown. Thus spindle position corresponds to nuclear position. Cytoplasmic (or astral) microtubules (cMTs) emanate from the SPBs (spindle pole bodies), the MT (microtubule)-organizing centres, and are highly dynamic. cMTs are essential for nuclear positioning by connecting the SPB, which is embedded in the nuclear envelope, with the cell cortex, thus promoting the generation of forces that move the nucleus towards and inside the bud neck. Two sequential processes, the Kar9 pathway and the dynein pathway, contribute to spindle positioning (Figure 1). Either pathway is dispensable for cell viability, whereas inactivation of both is lethal . Kar9 is localized at the SPB and is translocated to MT plus ends through interaction with the plus end-directed motor Kip2 and the MT-associated Bim1 protein [8,9]. The Kar9/Bim1 complex guides MTs along polarized actin cables into the bud by interacting with the type V myosin Myo2 . Through these interactions, the Kar9 pathway promotes capture of cMTs with the bud cortex primarily prior to anaphase (Figure 1A). The second pathway of spindle positioning requires the minus end-directed motor dynein  (Figure 1B) that associates with the cortical anchor Num1 [12,13]. Targeting of dynein to MT plus ends requires the Bik1 plus end-tracking protein , which in turn binds MT ends through Kip2 . The dynein pathway acts predominantly during anaphase and might contribute to the cMT capture by the bud tip through sliding plus-ends of cMTs along the cortex (reviewed in ). These complicated interactions generate forces that place the nucleus at the bud neck and orientate the mitotic spindle with respect to the division axis.
The two pathways of budding yeast spindle positioning
Asymmetry of MT-organizing centres
The accuracy of spindle positioning is partly ensured by an asymmetry of spindle poles. In vertebrate cells, the old centriole migrates to the site of the cytokinetic furrow, indicating the existence of a specific pattern of centrosome inheritance . In C. elegans, some proteins are specifically recruited to one spindle pole [18,19], whereas in Drosophila germline stem cells, the mother centrosome is segregated into the cell that maintains stem cell identity . Similarly, in budding yeast, the old SPB is inherited by the daughter cell, while the newly synthesized SPB stays in the mother cell . SPB asymmetry is established at least at three levels: (i) the activity of S-phase CDK (cyclin-dependent kinase) transiently inhibits MT nucleation from the new SPB until formation of the bipolar spindle ; (ii) Kar9 localization is restricted exclusively to the bud-directed SPB [9,23]; (iii) dynein is also asymmetrically localized on the SPB moving towards the bud, but only in metaphase [24,25]. Transient restriction of astral MT formation from the old SPB helps to target this spindle pole to the bud. In addition, Kar9 presence only on the old SPB allows it to be oriented towards the bud by connecting cMT ends with cortical Myo2. Although the mechanisms that generate this asymmetry are not completely unravelled, Kar9 phosphorylation by CDKs is required to restrict Kar9 localization to the daughter-bound SPB and MTs, as well as for proper spindle positioning [9,23]. Bik1 is also involved in maintaining Kar9 asymmetry by favouring its phosphorylation .
Similarly to Kar9, asymmetric distribution of dynein at spindle poles in metaphase is thought to be required for spindle positioning. Dynein asymmetry involves the SPB component Cnm67, several protein kinases localized at the bud neck and mitotic CDKs . However, different sets of mitotic CDKs are involved in Kar9 and in dynein asymmetry: whereas the former depends on the Clb3 and Clb4 cyclins [23,27], the latter requires Clb1 and Clb2 .
Homologues of the above proteins regulate asymmetric cell division in higher eukaryotes. The dynein complex is involved in proper spindle positioning in Drosophila neuroblasts . Kar9 shares limited homology with APC (adenomatous polyposis coli), an MT-binding and -regulating protein that is in turn modulated by a protein complex involved in polarity establishment in mammals [28–30]. APC is required for control of spindle orientation in Drosophila stem cells to drive asymmetric cell division . EB1, the counterpart of budding yeast Bim1, is required in Drosophila for proper spindle positioning . Finally, cyclinA- and B-dependent CDKs regulate asymmetric cell division by modulating the localization of asymmetric determinants (reviewed in ). Altogether, evolutionary conservation of these proteins indicate that molecular mechanisms of spindle polarity and positioning might be conserved from yeast to humans.
The spindle position checkpoint
In case of spindle orientation defects, in budding yeast mitotic exit and cytokinesis are delayed to provide the time necessary for error correction [34–37] and this is crucial to prevent unbalanced chromosome partitioning. The SPOC (spindle position checkpoint) is responsible for this cell cycle delay by inhibiting the MEN (mitotic exit network) signal transduction cascade. The MEN involves several factors, like the small GTPase Tem1 and several protein kinases (Cdc15, Dbf2 associated to its activator Mob1 and the polo kinase Cdc5), which ultimately promote the release from the nucleolus and activation of a key phosphatase (Cdc14, Figure 2). Cdc14 is in turn essential for mitotic exit and cytokinesis through inactivation of mitotic CDKs and dephosphorylation of their targets (reviewed in ). A pathway organized similarly to the MEN is the fission yeast SIN (septation initiation network), which is required for cytokinesis . In addition, several MEN and SIN components can be found in higher eukaryotic systems, from plants to human, raising the possibility that similar pathways might also exist in multicellular eukaryotes to survey the correct patterns of asymmetric cell division during development.
Tem1 is the direct target of the SPOC and its activity is finely regulated in space and time (Figure 2, reviewed in [38,40,41]). The two component GAP (GTPase-activating protein) Bub2/Bfa1 binds Tem1 and inhibits it by stimulating GTP hydrolysis [42,43]. The GAP activity resides on Bub2 [42,43], which carries a GAP TBC (Tre-2, Bub2 and Cdc16 ) domain, whereas Bfa1 is required for Bub2 interaction with Tem1 . Circumstantial evidence suggests that Bfa1 might prevent Tem1 activation also independently of Bub2: Bfa1 interferes with Tem1 binding to its effector kinase Cdc15 and its overexpression inhibits mitotic exit independently of Bub2 . Since Bfa1 is able to freeze Tem1 in its GTP-bound form by preventing GTP dissociation and hydrolysis [42,43], it might act as a GDI (guanine-nucleotide dissociation inhibitor), which regulate cognate GTPases by blocking GTP/GDP exchange . Whether Bfa1 is indeed a GDI for Tem1 remains an open and important question, as this activity could be physiologically relevant to inhibit Tem1 in response to spindle mispositioning.
Tem1 activation is somehow stimulated by the putative GEF (guanine-nucleotide-exchange factor) Lte1 , but the molecular mechanism at the basis of activation is still unknown. Lte1 is necessary for mitotic exit only at low temperatures  or when mitotic exit is partially impaired, such as in mutants defective in Cdc14 early anaphase release from the nucleolus . In addition, its GEF domain appears to be dispensable for Tem1 activation, although it is required for Lte1 localization to the bud cortex . Finally, mitotic exit can occur on schedule in the absence of Lte1 . Recent data indicate that the formin Bud6 and the kinesin Kip2, both involved in MT cortical capture, are part of the SPOC and may signal the absence of the spindle from the bud neck to keep Lte1 inactive. This inhibitory signal would be relieved when the spindle crosses the bud neck and MT-cortex interactions are lost .
The subcellular localization of Tem1 and its regulators is finely tuned during the cell cycle: in G1, Tem1, Bub2 and Bfa1 are present on the single SPB, whereas soon after SPB duplication and up to metaphase they are found on both SPBs (Figure 2B, [37,52]). When the spindle is properly oriented and after the onset of anaphase, Tem1 accumulates at higher levels on the SPB that is present in the bud in an Lte1-dependent manner , while Bub2/Bfa1 are displaced from the mother-bound SPB [37,52]. Since Lte1 is present only in the bud [35,37,52], these observations led to an attractive model for the SPOC: as long as the mitotic spindle is not correctly positioned, the GAP Bub2/Bfa1 keeps Tem1 inactive. Passage of one spindle pole through the bud neck signals that nuclear division has taken place correctly and Tem1 is activated thanks to exposure to its activator Lte1, which is present only in the bud, and inhibition of Bub2/Bfa1 through phosphorylation by the Polo-like kinase Cdc5 [35,37,53]. This would implicate that the pool of Tem1 residing at the bud-directed SPB is mainly responsible for triggering MEN activation, mitotic exit and cytokinesis. However, the pool of Tem1 localized on the mother-bound SPB turned out recently to contribute to timely mitotic exit, as persistence of Bub2/Bfa1 at this SPB during anaphase prevents mitotic exit in mutants where Tem1 activity is partially impaired [41,42]. Accordingly, the Bub2/Bfa1 complex is retained on both SPBs of anaphase cells with mispositioned spindles (Figure 2A, [21,52]). Furthermore, the protein kinase Kin4, which is also required for the SPOC [54,55] by antagonizing the inhibitory phosphorylation of Bub2/Bfa1 by Cdc5 , localizes to the mother-bound SPB and binds to both SPBs in case of spindle misalignment . Interestingly, SPB localization of Kin4 is mediated by the SPB component Spc72 that directly interacts with the γ-tubulin complex, thus linking a SPOC component with cytoplasmic MTs .
Disappearance of Bub2/Bfa1 from the mother-bound SPB during anaphase requires Bub2 GAP activity and a functional septin ring . The latter is localized at the bud neck and is required to maintain cell polarity by specifying a boundary between mother and daughter cell . The septin ring is also required for Lte1 localization in the bud: lack of septins causes mislocalization of Lte1 to the mother cell and abolishes the SPOC . Indeed, during telophase of unperturbed cell cycles, Lte1 asymmetry is lost and the protein diffuses from the bud cortex to the cytoplasm of both mother cell and bud (Figure 2B, [35,37]), thus perhaps contributing to Tem1 activation in the mother cell. Altogether, these data suggest that key regulatory events preventing the MEN upon spindle mispositioning take place within the mother cell. In addition, the involvement of the septin ring in this process would provide an explanation for the finding that mitotic exit is signalled by passage of one SPB through the bud neck .
In conclusion, the SPOC controls Tem1 activation in time and space by different mechanisms, thus allowing the cells to progress safely out of mitosis, to divide and to generate daughter cells with a single and complete copy of the genome, preventing cell death and the formation of cells with unbalanced ploidies. Since many proteins involved in mitotic spindle positioning and MEN components are conserved from yeast to humans, one of the future challenges will be to discover whether safety devices like the SPOC could operate in other cell types that divide asymmetrically.
Mechanics and Control of Cytokinesis: Biochemical Society Focused Meeting held at Royal College of Surgeons, Edinburgh, U.K., 9–12 January 2008. Organized and Edited by Gwyn Gould (Glasgow, U.K.) and Iain Hagan (Manchester, U.K.).
adenomatous polyposis coli
cell division cycle
guanine-nucleotide dissociation inhibitor
mitotic exit network
septation initiation network
spindle pole body
spindle position checkpoint
We thank Maria Pia Longhese and Giovanna Lucchini for comments on the manuscript. Work in S.P.'s laboratory is funded by grants from Associazione Italiana Ricerca sul Cancro, PRIN (Progetti di Ricerca di Interesse Nazionale) and Telethon. M.V. is supported by a Fondazione Fratelli Confalonieri Fellowship.