Michele H. Jones

Chemical Genetics Reveals a Role for Mps1 Kinase in Kinetochore Attachment during Mitosis

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.

A Cell Cycle Phosphoproteome of the Yeast Centrosome

Phosphorylation of the yeast centrosome reveals sites of regulation and predicts complex regulation of mammalian centrosomes. Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)–directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle

During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.

Centrosome Duplication: Is Asymmetry the Clue?

The structure of the yeast Sfi1-centrin complex, and its asymmetric position within the yeast centrosome, suggest a model for the initiation of centrosome duplication and provides a target for licensing this event.

Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.

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

A model of the core of the yeast spindle pole body (SPB) was created by a Bayesian modeling approach that integrated a diverse data set of biophysical, biochemical, and genetic information. The model led to a proposed pathway for the assembly of Spc110, a protein related to pericentrin, and a mechanism for how calmodulin strengthens the SPB during mitosis.

Cdk1 (Cdc28) Phosphorylation of Gamma-Tubulin Couples Spindle Microtubule Dynamics to the Metaphase-Anaphase Transition

γ-Tubulin is an evolutionarily conserved nucleator of microtubules. γ-Tubulin is a phospho-protein and the phosphorylation state of a conserved residue in the C-terminus (Y445) contributes to normal spindle function and actin-microtubule coupling in budding yeast. Here we report that γ-tubulin is phosphorylated at S360, a Cdk1/Cdc28 site. Phosphorylation of S360 in vivo was identified in a global analysis of the phosphoproteome of the spindle pole body. We confirmed Cdc28-Clb2 could phosphorylate S360 by in vitro kinase assay and peptide identification by mass spectrometry, and in vivo using two dimensional-PAGE. A phospho-mimetic mutation (tub4-S360D) causes mitotic delay but does not inhibit recruitment of the γ-tubulin complex (reported by GRIP Spc97-EGFP) to spindle poles. Cytoplasmic microtubule function is normal in tub4-S360D cells but spindle microtubule function is altered. High-resolution analysis of spindle dynamics revealed fluctuations in length in metaphase and anaphase spindles. The velocities of spindle elongation in anaphase were similar in S360D and wild-type spindles, but the initial phase of rapid elongation in anaphase is prolonged in the S360D mutant. We propose that S360 phosphorylation of γ-tubulin by Cdk1 may play an important role in the control of spindle microtubule dynamics during the metaphase-anaphase transition.