A mutation in budding yeast BRR6 affecting nuclear envelope insertion of the spindle pole body.

BRR6 and BRL1 are two paralogs that encode transmembrane proteins of the nuclear envelope (NE) involved in membrane fluidity and nuclear pore complex biogenesis in organisms that undergo a closed mitosis. We show that mutation of a conserved cysteine in the intralumenal domain of Saccharomyces cerevisiae Brr6p results in a novel temperature sensitive allele, brr6-Y100H, that arrests growth due to defects in spindle formation. Analysis of brr6-Y100H cells by electron tomography and Brr6p localization by super-resolution imaging supports the idea that Brr6p is involved in insertion of the newly duplicated spindle pole body into the NE.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

Binding of Drosophila Polo kinase to its regulator Matrimony is noncanonical and involves two separate functional domains.

Drosophila melanogaster Polo kinase physically interacts with, and is repressed by, the Matrimony (Mtrm) protein during oogenesis. Females heterozygous for a deletion of the mtrm gene display defects in chromosome segregation at meiosis I. However, a complete absence of Mtrm results in both meiotic catastrophe and female sterility. We show that three phosphorylated residues in an N-terminal region in Mtrm are required for Mtrm::Polo binding. However, this binding is noncanonical; it does not require either a complete S-pS/pT-P motif in Mtrm or key residues in the Polo-box domain of Polo that allow Polo to bind phosphorylated substrates. By using fluorescence cross-correlation spectroscopy to characterize the Mtrm::Polo interaction in vivo, we show that a sterile α-motif (SAM) domain located at the C terminus of Mtrm increases the stability of Mtrm::Polo binding. Although Mtrm's C-terminal SAM domain is not required to rescue the chromosome segregation defects observed in mtrm/+ females, it is essential to prevent both meiotic catastrophe and the female sterility observed in mtrm/mtrm females. We propose that Polo's interaction with the cluster of phosphorylated residues alone is sufficient to rescue the meiosis I defect. However, the strengthening of Mtrm::Polo binding mediated by the SAM domain is necessary to prevent meiotic catastrophe and ensure female fertility. Characterization of the Mtrm::Polo interaction, as well as that of other Polo regulators, may assist in the design of a new class of Polo inhibitors to be used as targeted anticancer therapeutic agents.

Eco1 is important for DNA damage repair in S. cerevisiae.

The cohesin network has an essential role in chromosome segregation, but also plays a role in DNA damage repair. Eco1 is an acetyltransferase that targets subunits of the cohesin complex and is involved in both the chromosome segregation and DNA damage repair roles of the network. Using budding yeast as a model system, we find that mutations in Eco1, including a genocopy of a human Roberts syndrome allele, do not cause gross defects in chromosome cohesion. We examined how mitotic and meiotic DNA damage repair is affected by mutations in Eco1. Strains containing mutations in Eco1 are sensitive to DNA damaging agents that cause double-strand breaks, such as X-rays and bleomycin. While meiotic crossing over is relatively unaffected in strains containing the Roberts mutation, reciprocal mitotic crossovers occur with extremely low frequency in this mutant background. Our results suggest that Eco1 promotes the reciprocal exchange of chromosome arms and maintenance of heterozygosity during mitosis.

Anaphase inactivation of the spindle checkpoint.

The spindle checkpoint delays cell cycle progression until microtubules attach each pair of sister chromosomes to opposite poles of the mitotic spindle. Following sister chromatid separation, however, the checkpoint ignores chromosomes whose kinetochores are attached to only one spindle pole, a state that activates the checkpoint prior to metaphase. We demonstrate that, in budding yeast, mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the checkpoint, leads to sustained inactivation of the spindle checkpoint. Mps1 protein abundance decreases in anaphase, and Mps1 is a target of the APC. Furthermore, expression of Mps1 in anaphase, or repression of the APC in anaphase, reactivates the spindle checkpoint. This APC-Mps1 feedback circuit allows cells to irreversibly inactivate the checkpoint during anaphase.