The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body.

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.

Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication.

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

The half-bridge component Kar1 promotes centrosome separation and duplication during budding yeast meiosis.

The budding yeast centrosome, often called the spindle pole body (SPB), nucleates microtubules for chromosome segregation during cell division. An appendage, called the half bridge, attaches to one side of the SPB and regulates SPB duplication and separation. Like DNA, the SPB is duplicated only once per cell cycle. During meiosis, however, after one round of DNA replication, two rounds of SPB duplication and separation are coupled with homologue segregation in meiosis I and sister-chromatid segregation in meiosis II. How SPB duplication and separation are regulated during meiosis remains to be elucidated, and whether regulation in meiosis differs from that in mitosis is unclear. Here we show that overproduction of the half-bridge component Kar1 leads to premature SPB separation during meiosis. Furthermore, excessive Kar1 induces SPB overduplication to form supernumerary SPBs, leading to chromosome missegregation and erroneous ascospore formation. Kar1--mediated SPB duplication bypasses the requirement of dephosphorylation of Sfi1, another half-bridge component previously identified as a licensing factor. Our results therefore reveal an unexpected role of Kar1 in licensing meiotic SPB duplication and suggest a unique mechanism of SPB regulation during budding yeast meiosis.

Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function.

Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes [known as spindle pole bodies (SPBs)] are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.

Molecular model of fission yeast centrosome assembly determined by superresolution imaging.

Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the Schizosaccharomyces pombe SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 S. pombe SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the S. pombe SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment.

Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes.

The spindle pole body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

Evaluation of the Dynamicity of Mitotic Exit Network and Spindle Position Checkpoint Components on Spindle Pole Bodies by Fluorescence Recovery After Photobleaching (FRAP).

Fluorescence recovery after photobleaching (FRAP) is a powerful technique to study in vivo binding and diffusion dynamics of fluorescently labeled proteins. In this chapter, we describe how to determine spindle pole body (SPB) binding dynamics of mitotic exit network (MEN) and spindle position checkpoint (SPOC) proteins using FRAP microscopy. Procedures presented here include the growth of the yeast cultures, sample preparation, image acquisition and analysis.

Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.

Most proteins of the Mitotic Exit Network (MEN) and their upstream regulators localize at spindle pole bodies (SPBs) at least in some stages of the cell cycle. Studying the SPB localization of MEN factors has been extremely useful to elucidate their biological roles, organize them in a hierarchical pathway, and define their dynamics under different conditions.Recruitment to SPBs of the small GTPase Tem1 and the downstream kinases Cdc15 and Mob1/Dbf2 is thought to be essential for Cdc14 activation and mitotic exit, while that of the upstream Tem1 regulators (the Kin4 kinase and the GTPase activating protein Bub2-Bfa1) is important for MEN inhibition upon spindle mispositioning. Here, we describe the detailed fluorescence microscopy procedures that we use in our lab to analyze the localization at SPBs of Mitotic Exit Network (MEN) components tagged with GFP or HA epitopes.

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.

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.

Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana.

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.