Summary Proper assembly of the kinetochore, a multi-protein complex that mediates attachment of centromere DNA to spindle microtubules on each chromosome, is required for faithful chromosome segregation. Each previously characterized member of the Mis12/Mtw1 protein family is part of an essential sub-complex in the kinetochore. In this work, we identify and characterize CaMTW1, which encodes the homolog of the human Mis12 protein in the pathogenic budding yeast Candida albicans. Subcellular localization and chromatin immunoprecipitation assays confirmed CaMtw1 is a kinetochore protein. CaMtw1 is essential for viability. CaMtw1-depleted cells and cells in which CaMtw1 was inactivated with a temperature-sensitive mutation had reduced viability, accumulated at the G2/M stage of the cell cycle, and exhibited increased chromosome missegregation. CaMtw1 depletion also affected spindle length and alignment. Interestingly, in C. albicans, CaMtw1 and the centromeric histone, CaCse4, influence each other for kinetochore localization. In addition, CaMtw1 is required for efficient kinetochore recruitment of another inner kinetochore protein, the CENP-C homolog, CaMif2. Mis12/Mtw1 proteins have well-established roles in the recruitment and maintenance of outer kinetochore proteins. We propose that Mis12/Mtw1 proteins also have important co-dependent interactions with inner kinetochore proteins and that these interactions may increase the fidelity of kinetochore formation.

Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore–microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore–microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species.A complete understanding of the complexities of the process of cellular differentiation requires a thorough analysis of the molecular events occurring during eukaryotic cell division. As an important part of this process, a cell has to ensure accurate segregation of duplicated chromosomes into its progeny cells. In eukaryotes, specific DNA sequences, and the factors that bind to them immediately after replication, partly dictate the state of chromatin. Apart from the genetic factors, many epigenetic phenomena also contribute to formation of specialized chromatin required for specific functions. One such specialized chromatin domain, the centromere (CEN)-kinetochore complex, plays a crucial role in high-fidelity chromosome segregation and has great implications for human health. A consequence of improper chromosome segregation is the abnormal chromosome numbers associated with most human cancers. For example, in human colorectal cancers, almost 85% of cells are aneuploid, with 60 to 90 chromosomes (128).The high-fidelity chromosome segregation that occurs during mitosis and meiosis requires a functional centromere, defined as the primary constriction on a chromosome. The centromere is a region where spindle fibers attach to ...

Quantitative knowledge of the recruitment of spindle assembly checkpoint (SAC) proteins by the kinetochore is essential to understanding the mechanisms that regulate protein recruitment and hence the strength of the SAC. Here this recruitment is quantified, and novel mechanisms are identified that strongly modulate SAC protein recruitment by the kinetochore.

The Spindle Assembly Checkpoint (SAC) maintains genome stability while enabling timely anaphase onset. To maintain genome stability, the SAC must be strong so that it delays cell division even if one chromosome is unattached, but for timely anaphase onset, it must be responsive to silencing mechanisms. How it meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of 'MELT' motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Spc105/KNL1 must contain many strong MELT motifs to prevent chromosome missegregation, but not too many, because this delays SAC silencing and anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for significantly improved chromosome segregation accuracy. We propose that the necessity of balancing SAC strength with responsiveness drives the evolutionary trend of MELT motif number amplification and degeneration of their functionally optimal amino acid sequence.