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Shugoshin proteins are evolutionarily conserved across eukaryotes, with some species-specific cellular functions, ensuring the fidelity of chromosome segregation. They act as adaptors at various subcellular locales to mediate several protein-protein interactions in a spatio-temporal manner. Here, we characterize shugoshin (Sgo1) in the human fungal pathogen Candida albicans. We observe that Sgo1 retains its centromeric localization and performs its conserved functions of regulating the sister chromatid biorientation, centromeric condensin localization, and maintenance of chromosomal passenger complex (CPC). We identify novel roles of Sgo1 as a spindle assembly checkpoint (SAC) component with functions in maintaining a prolonged SAC response by retaining Mad2 and Bub1 at the kinetochores in response to improper kinetochore-microtubule attachments. Strikingly, we discover the in vivo localization of Sgo1 along the length of the mitotic spindle. Our results indicate that Sgo1 performs a hitherto unknown function of facilitating timely disassembly of the mitotic spindle in C. albicans. To summarize, this study unravels a unique functional adaptation of shugoshin in maintaining genomic stability.

Shugoshin proteins are evolutionary conserved across eukaryotes with some species-specific cellular functions ensuring the fidelity of chromosome segregation. Shugoshin being present at various subcellular locales, acts as an adaptor to mediate various protein-protein interactions in a spatio-temporal manner. Here, we characterize shugoshin (Sgo1) in the human fungal pathogen,Candida albicans. Interestingly, we discover a novelin vivolocalization of Sgo1 along the length of the mitotic spindle. Further, Sgo1 performs a hitherto unknown function of facilitating timely disassembly of spindle in this organism. We observe that Sgo1 retains its centromeric localization and performs its conserved functions that include regulating the centromeric condensin localization, chromosome passenger complex (CPC) maintenance and sister chromatid biorientation. We identify novel roles of Sgo1 as a spindle assembly checkpoint (SAC) component with functions in maintaining the SAC proteins, Mad2 and Bub1, at the kinetochores, in response to faulty kinetochore-microtubule attachments. These findings provide an excellent evidence of the functional rewiring of shugoshin in maintaining genomic stability.

Chromosome transmission fidelity during mitosis is of critical importance for the fitness of an organism, as mistakes will lead to aneuploidy, which has a causative role in numerous severe diseases. Proper segregation of chromosomes depends on interdependent processes at the microtubule-kinetochore interface and the spindle assembly checkpoint. Here we report the discovery of a new element essential for chromosome transmission fidelity that implicates inositol pyrophosphates (IPPs) as playing a key role in this process. The protein is Asp1, the Schizosaccharomyces pombe member of the highly conserved Vip1 family. Vip1 enzymes are bifunctional: they consist of an IPP-generating kinase domain and a pyrophosphatase domain that uses such IPPs as substrates. We show that Asp1 kinase function is required for bipolar spindle formation. The absence of Asp1-generated IPPs resulted in errors in sister chromatid biorientation, a prolonged checkpoint-controlled delay of anaphase onset, and chromosome missegregation. Remarkably, expression of Asp1 variants that generated higher-than-wild-type levels of IPPs led to a faster-than-wild-type entry into anaphase A without an increase in chromosome missegregation. In fact, the chromosome transmission fidelity of a nonessential chromosome was enhanced with increased cellular IPPs. Thus, we identified an element that optimized the wild-type chromosome transmission process.

Summary Background The KMN network, a ten-subunit protein complex, mediates the interaction of kinetochores with spindle microtubules and recruits spindle assembly checkpoint (SAC) constituents to halt cells in mitosis until attainment of sister chromatid biorientation. Two types of motifs in the KMN subunit Knl1 interact with SAC proteins. Lys-Ile (KI) motifs, found in vertebrates, interact with the TPR motifs of Bub1 and BubR1. Met-Glu-Leu-Thr (MELT) repeats, ubiquitous in evolution, recruit the Bub3/Bub1 complex in a phosphorylation-dependent manner. The exact contributions of KI and MELT motifs to SAC signaling and chromosome alignment are unclear. Results We report here that KI motifs cooperate strongly with the neighboring single MELT motif in the N-terminal 250 residues (Knl1 1–250 ) of human Knl1 to seed a comprehensive assembly of SAC proteins. In cells depleted of endogenous Knl1, kinetochore-targeted Knl1 1–250 suffices to restore SAC and chromosome alignment. Individual MELT repeats outside of Knl1 1–250 , which lack flanking KI motifs, establish qualitatively similar sets of interactions, but less efficiently. Conclusions MELT sequences on Knl1 emerge from our analysis as the platforms on which SAC complexes become assembled. Our results show that KI motifs are enhancers of MELT function in assembling SAC signaling complexes, and that they might have evolved to limit the expansion of MELT motifs by providing a more robust mechanism of SAC signaling around a single MELT. We shed light on the mechanism of Bub1 and BubR1 recruitment and identify crucial questions for future studies.

The spindle assembly checkpoint (SAC) is a surveillance mechanism contributing to the preservation of genomic stability by monitoring the microtubule attachment to, and/or the tension status of, each kinetochore during mitosis. The SAC halts metaphase to anaphase transition in the presence of unattached and/or untensed kinetochore(s) by releasing the mitotic checkpoint complex (MCC) from these improperly-oriented kinetochores to inhibit the anaphase-promoting complex/cyclosome (APC/C). The reversible phosphorylation of a variety of substrates at the kinetochore by antagonistic kinases and phosphatases is one major signaling mechanism for promptly turning on or turning off the SAC. In such a complex network, some kinases act at the apex of the SAC cascade by either generating (monopolar spindle 1, MPS1/TTK and likely polo-like kinase 1, PLK1), or contributing to generate (Aurora kinase B) kinetochore phospho-docking sites for the hierarchical recruitment of the SAC proteins. Aurora kinase B, MPS1 and budding uninhibited by benzimidazoles 1 (BUB1) also promote sister chromatid biorientation by modulating kinetochore microtubule stability. Moreover, MPS1, BUB1, and PLK1 seem to play key roles in APC/C inhibition by mechanisms dependent and/or independent on MCC assembly. The protein phosphatase 1 and 2A (PP1 and PP2A) are recruited to kinetochores to oppose kinase activity. These phosphatases reverse the phosphorylation of kinetochore targets promoting the microtubule attachment stabilization, sister kinetochore biorientation and SAC silencing. The kinase-phosphatase network is crucial as it renders the SAC a dynamic, graded-signaling, high responsive, and robust process thereby ensuring timely anaphase onset and preventing the generation of proneoplastic aneuploidy.