Spindle pole bodies function as signal amplifiers in the Mitotic Exit Network.

The Mitotic Exit Network (MEN), a budding yeast Ras-like signal transduction cascade, translates nuclear position into a signal to exit from mitosis. Here we describe how scaffolding the MEN onto spindle pole bodies (SPB-centrosome equivalent) allows the MEN to couple the final stages of mitosis to spindle position. Through the quantitative analysis of the localization of MEN components, we determined the relative importance of MEN signaling from the SPB that is delivered into the daughter cell (dSPB) during anaphase and the SPB that remains in the mother cell. Movement of half of the nucleus into the bud during anaphase causes the active form of the MEN GTPase Tem1 to accumulate at the dSPB. In response to Tem1's activity at the dSPB, the MEN kinase cascade, which functions downstream of Tem1, accumulates at both SPBs. This localization to both SPBs serves an important role in promoting efficient exit from mitosis. Cells that harbor only one SPB delay exit from mitosis. We propose that MEN signaling is initiated by Tem1 at the dSPB and that association of the downstream MEN kinases with both SPBs serves to amplify MEN signaling, enabling the timely exit from mitosis.

Asymmetric Segregation of Aged Spindle Pole Bodies During Cell Division: Mechanisms and Relevance Beyond Budding Yeast?

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non-random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB-inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre-existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non-random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY.

The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling.

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

Direct measurement of the strength of microtubule attachment to yeast centrosomes.

Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubule-centrosome attachments is unknown, and the possibility that mechanical force might regulate centrosome function has scarcely been explored. To uncover how centrosomes sustain and regulate force, we purified SPBs from budding yeast and used laser trapping to manipulate single attached microtubules in vitro. Our experiments reveal that SPB-microtubule attachments are extraordinarily strong, rupturing at forces approximately fourfold higher than kinetochore attachments under identical loading conditions. Furthermore, removal of the calmodulin-binding site from the SPB component Spc110 weakens SPB-microtubule attachment in vitro and sensitizes cells to increased SPB stress in vivo. These observations show that calmodulin binding contributes to SPB mechanical integrity and suggest that its removal may cause pole delamination and mitotic failure when spindle forces are elevated. We propose that the very high strength of SPB-microtubule attachments may be important for spindle integrity in mitotic cells so that tensile forces generated at kinetochores do not cause microtubule detachment and delamination at SPBs.

Mitotic Nuclear Envelope Breakdown and Spindle Nucleation Are Controlled by Interphase Contacts between Centromeres and the Nuclear Envelope.

Faithful genome propagation requires coordination between nuclear envelope (NE) breakdown, spindle formation, and chromosomal events. The conserved linker of nucleoskeleton and cytoskeleton (LINC) complex connects fission yeast centromeres and the centrosome, across the NE, during interphase. During meiosis, LINC connects the centrosome with telomeres rather than centromeres. We previously showed that loss of telomere-LINC contacts compromises meiotic spindle formation. Here, we define the precise events regulated by telomere-LINC contacts and address the analogous possibility that centromeres regulate mitotic spindle formation. We develop conditionally inactivated LINC complexes in which the conserved SUN-domain protein Sad1 remains stable but severs interphase centromere-LINC contacts. Strikingly, the loss of such contacts abolishes spindle formation. We pinpoint the defect to a failure in the partial NE breakdown required for centrosome insertion into the NE, a step analogous to mammalian NE breakdown. Thus, interphase chromosome-LINC contacts constitute a cell-cycle control device linking nucleoplasmic and cytoplasmic events.

Duplication of the Yeast Spindle Pole Body Once per Cell Cycle.

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. Centrosomes and SPBs duplicate exactly once per cell cycle by mechanisms that use the mother structure as a platform for the assembly of the daughter. The conserved Sfi1 and centrin proteins are essential components of the SPB duplication process. Sfi1 is an elongated molecule that has, in its center, 20 to 23 binding sites for the Ca(2+)-binding protein centrin. In the yeastSaccharomyces cerevisiae, all Sfi1 N termini are in contact with the mother SPB whereas the free C termini are distal to it. During S phase and early mitosis, cyclin-dependent kinase 1 (Cdk1) phosphorylation of mainly serine residues in the Sfi1 C termini blocks the initiation of SPB duplication ("off" state). Upon anaphase onset, the phosphatase Cdc14 dephosphorylates Sfi1 ("on" state) to promote antiparallel and shifted incorporation of cytoplasmic Sfi1 molecules into the half-bridge layer, which thereby elongates into the bridge. The Sfi1 C termini of the two Sfi1 layers localize in the bridge center, whereas the N termini of the newly assembled Sfi1 molecules are distal to the mother SPB. These free Sfi1 N termini then assemble the new SPB in G1phase. Recruitment of Sfi1 molecules into the anaphase SPB and bridge formation were also observed inSchizosaccharomyces pombe, suggesting that the Sfi1 bridge cycle is conserved between the two organisms. Thus, restricting SPB duplication to one event per cell cycle requires only an oscillation between Cdk1 kinase and Cdc14 phosphatase activities. This clockwork regulates the "on"/"off" state of the Sfi1-centrin receiver.

Monitoring SPB biogenesis in fission yeast with high resolution and quantitative fluorescent microscopy.

Like centrosomes, yeast spindle pole bodies (SPBs) undergo a tightly controlled duplication cycle in order to restrict their number to one or two per cell and promote the assembly of a bipolar spindle at mitotic entry. This conservative duplication cycle is tightly coordinated with cell cycle progression although the mechanisms that ensure this coordination remain largely unknown. In this chapter, we describe simple high resolution microscopy- and quantitative light microscopy-based methods that allow to monitor SPB biogenesis in fission yeast and may be useful to study the molecular pathways controlling the successive phases of the duplication cycle.

Evolution of zygomycetous spindle pole bodies: Evidence from Coemansia reversa mitosis.

PREMISE OF THE STUDY: The earliest eukaryotes were likely flagellates with a centriole that nucleates the centrosome, the microtubule-organizing center (MTOC) for nuclear division. The MTOC in higher fungi, which lack flagella, is the spindle pole body (SPB). Can we detect stages in centrosome evolution leading to the diversity of SPB forms observed in terrestrial fungi? Zygomycetous fungi, which consist of saprobes, symbionts, and parasites of animals and plants, are critical in answering the question, but nuclear division has been studied in only two of six clades. METHODS: Ultrastructure of mitosis was studied in Coemansia reversa (Kickxellomycotina) germlings using cryofixation or chemical fixation. Character evolution was assessed by parsimony analysis, using a phylogenetic tree assembled from multigene analyses. KEY RESULTS: At interphase the SPB consisted of two components: a cytoplasmic, electron-dense sphere containing a cylindrical structure with microtubules oriented nearly perpendicular to the nucleus and an intranuclear component appressed to the nuclear envelope. Markham's rotation was used to reinforce the image of the cylindrical structure and determine the probable number of microtubules as nine. The SPB duplicated early in mitosis and separated on the intact nuclear envelope. Nuclear division appears to be intranuclear with spindle and kinetochore microtubules interspersed with condensed chromatin. CONCLUSIONS: This is the sixth type of zygomycetous SPB, and the third type that suggests a modified centriolar component. Coemansia reversa retains SPB character states from an ancestral centriole intermediate between those of fungi with motile cells and other zygomycetous fungi and Dikarya.

Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast.

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.

Sharpening the anaphase switch.

The segregation of sister chromatids during mitosis is one of the most easily visualized, yet most remarkable, events during the life cycle of a cell. The accuracy of this process is essential to maintain ploidy during cell duplication. Over the past 20 years, substantial progress has been made in identifying components of both the kinetochore and the mitotic spindle that generate the force to move mitotic chromosomes. Additionally, we now have a reasonable, albeit incomplete, understanding of the molecular and biochemical events that are involved in establishing and dissolving sister-chromatid cohesion. However, it is less well-understood how this dissolution of cohesion occurs synchronously on all chromosomes at the onset of anaphase. At the centre of the action is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that, in association with its activator cell-division cycle protein 20 homologue (Cdc20), is responsible for the destruction of securin. This leads to the activation of separase, a specialized protease that cleaves the kleisin-subunit of the cohesin complex, to relieve cohesion between sister chromatids. APC/C-Cdc20 is also responsible for the destruction of cyclin B and therefore inactivation of the cyclin B-cyclin-dependent kinase 1 (Cdk1). This latter event induces a change in the microtubule dynamics that results in the movement of sister chromatids to spindle poles (anaphase A), spindle elongation (anaphase B) and the onset of cytokinesis. In the present paper, we review the emerging evidence that multiple, spatially and temporally regulated feedback loops ensure anaphase onset is rapid, co-ordinated and irreversible.

Exploring the evolutionary history of centrosomes.

The centrosome is the main organizer of the microtubule cytoskeleton in animals, higher fungi and several other eukaryotic lineages. Centrosomes are usually located at the centre of cell in tight association with the nuclear envelope and duplicate at each cell cycle. Despite a great structural diversity between the different types of centrosomes, they are functionally equivalent and share at least some of their molecular components. In this paper, we explore the evolutionary origin of the different centrosomes, in an attempt to understand whether they are derived from an ancestral centrosome or evolved independently from the motile apparatus of distinct flagellated ancestors. We then discuss the evolution of centrosome structure and function within the animal lineage.

Lessons from yeast: the spindle pole body and the centrosome.

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.

Centrosomes as signalling centres.

Centrosomes-as well as the related spindle pole bodies (SPBs) of yeast-have been extensively studied from the perspective of their microtubule-organizing roles. Moreover, the biogenesis and duplication of these organelles have been the subject of much attention, and the importance of centrosomes and the centriole-ciliary apparatus for human disease is well recognized. Much less developed is our understanding of another facet of centrosomes and SPBs, namely their possible role as signalling centres. Yet, many signalling components, including kinases and phosphatases, have been associated with centrosomes and spindle poles, giving rise to the hypothesis that these organelles might serve as hubs for the integration and coordination of signalling pathways. In this review, we discuss a number of selected studies that bear on this notion. We cover different processes (cell cycle control, development, DNA damage response) and organisms (yeast, invertebrates and vertebrates), but have made no attempt to be comprehensive. This field is still young and although the concept of centrosomes and SPBs as signalling centres is attractive, it remains primarily a concept-in need of further scrutiny. We hope that this review will stimulate thought and experimentation.

Centrosome duplication: suspending a license by phosphorylating a template.

The phosphorylation status of Sfi1, a structural component of the yeast centrosome, governs the centrosome duplication cycle, raising the possibility that licensing of centrosome duplication occurs by modulating Sfi1, which potentially acts as a template for a new centrosome.

Molecular mechanisms that restrict yeast centrosome duplication to one event per cell cycle.

BACKGROUND: The spindle pole body (SPB) of budding yeast is the functional equivalent of the mammalian centrosome. Like the centrosome, the SPB duplicates once per cell cycle. The new SPB assembles adjacent to the mother SPB at a substructure called the bridge. The half-bridge, the bridge precursor, is a one-sided extension of the SPB central plaque layered on both sides of the nuclear envelope. Parallel Sfi1 molecules longitudinally span the half-bridge with their N termini embedded in the SPB central plaque, whereas their C termini mark the half-bridge distal end. In early G1, half-bridge elongation by antiparallel C-to-C dimerization of Sfi1 exposes free N-Sfi1 where the new SPB assembles. After SPB duplication, the dimerized Sfi1 is severed to allow spindle formation and SPB reduplication. RESULTS: We show that Sfi1 C-terminal domain harbors phosphorylation sites for Cdk1 and the polo-like kinase Cdc5. Cdk1 and, to a lesser extent, Cdc5 inhibit SPB duplication as phosphomimetic sfi1 mutations lead to metaphase cells with a single SPB. In contrast, phosphoinhibitory sfi1 mutations in Cdk1 sites are lethal because cells fail to sever the bridge after SPB duplication. Moreover, Cdc14 dephosphorylates C-Sfi1 to prepare it for a new round of duplication, and the kinase Mps1 promotes Sfi1 extension in G1. CONCLUSIONS: Positive (Cdc14) and negative (Cdk1 and Cdc5) SPB duplication signals are integrated at the level of the half-bridge component Sfi1. In addition, Mps1 activates Sfi1 duplication. Fluctuating activities of these regulators ensure one SPB duplication event per cell cycle.