Mitotic checkpoint gene expression is tuned by codon usage bias.

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+ , their short mRNA half-lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1+ mRNA has a short half-life despite a higher frequency of optimal codons, and despite the lack of known RNA-destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

Mutation and selection explain why many eukaryotic centromeric DNA sequences are often A + T rich.

We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (Ne) we also show that the relative A + T content of centromeric DNA scales with Ne across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.

Mps1 checks up on chromosome attachment.

The protein kinase Mps1, a crucial regulator of the spindle-assembly checkpoint, now turns out to be essential for correcting errors in chromosome attachment (Jelluma et al., 2008). Mps1 exerts this effect by regulating the activity of the Aurora B kinase through phosphorylation of its partner protein Borealin.

Robust Ordering of Anaphase Events by Adaptive Thresholds and Competing Degradation Pathways.

The splitting of chromosomes in anaphase and their delivery into the daughter cells needs to be accurately executed to maintain genome stability. Chromosome splitting requires the degradation of securin, whereas the distribution of the chromosomes into the daughter cells requires the degradation of cyclin B. We show that cells encounter and tolerate variations in the abundance of securin or cyclin B. This makes the concurrent onset of securin and cyclin B degradation insufficient to guarantee that early anaphase events occur in the correct order. We uncover that the timing of chromosome splitting is not determined by reaching a fixed securin level, but that this level adapts to the securin degradation kinetics. In conjunction with securin and cyclin B competing for degradation during anaphase, this provides robustness to the temporal order of anaphase events. Our work reveals how parallel cell-cycle pathways can be temporally coordinated despite variability in protein concentrations.

Implications of alternative routes to APC/C inhibition by the mitotic checkpoint complex.

The mitotic checkpoint (also called spindle assembly checkpoint) is a signaling pathway that ensures faithful chromosome segregation. Mitotic checkpoint proteins inhibit the anaphase-promoting complex (APC/C) and its activator Cdc20 to prevent precocious anaphase. Checkpoint signaling leads to a complex of APC/C, Cdc20, and checkpoint proteins, in which the APC/C is inactive. In principle, this final product of the mitotic checkpoint can be obtained via different pathways, whose relevance still needs to be fully ascertained experimentally. Here, we use mathematical models to compare the implications on checkpoint response of the possible pathways leading to APC/C inhibition. We identify a previously unrecognized funneling effect for Cdc20, which favors Cdc20 incorporation into the inhibitory complex and therefore promotes checkpoint activity. Furthermore, we find that the presence or absence of one specific assembly reaction determines whether the checkpoint remains functional at elevated levels of Cdc20, which can occur in cancer cells. Our results reveal the inhibitory logics behind checkpoint activity, predict checkpoint efficiency in perturbed situations, and could inform molecular strategies to treat malignancies that exhibit Cdc20 overexpression.

MEMO: multi-experiment mixture model analysis of censored data.

MOTIVATION: The statistical analysis of single-cell data is a challenge in cell biological studies. Tailored statistical models and computational methods are required to resolve the subpopulation structure, i.e. to correctly identify and characterize subpopulations. These approaches also support the unraveling of sources of cell-to-cell variability. Finite mixture models have shown promise, but the available approaches are ill suited to the simultaneous consideration of data from multiple experimental conditions and to censored data. The prevalence and relevance of single-cell data and the lack of suitable computational analytics make automated methods, that are able to deal with the requirements posed by these data, necessary. RESULTS: We present MEMO, a flexible mixture modeling framework that enables the simultaneous, automated analysis of censored and uncensored data acquired under multiple experimental conditions. MEMO is based on maximum-likelihood inference and allows for testing competing hypotheses. MEMO can be applied to a variety of different single-cell data types. We demonstrate the advantages of MEMO by analyzing right and interval censored single-cell microscopy data. Our results show that an examination of censoring and the simultaneous consideration of different experimental conditions are necessary to reveal biologically meaningful subpopulation structures. MEMO allows for a stringent analysis of single-cell data and enables researchers to avoid misinterpretation of censored data. Therefore, MEMO is a valuable asset for all fields that infer the characteristics of populations by looking at single individuals such as cell biology and medicine. AVAILABILITY AND IMPLEMENTATION: MEMO is implemented in MATLAB and freely available via github (https://github.com/MEMO-toolbox/MEMO). CONTACTS: eva-maria.geissen@ist.uni-stuttgart.de or nicole.radde@ist.uni-stuttgart.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mad1 contribution to spindle assembly checkpoint signalling goes beyond presenting Mad2 at kinetochores.

The spindle assembly checkpoint inhibits anaphase until all chromosomes have become attached to the mitotic spindle. A complex between the checkpoint proteins Mad1 and Mad2 provides a platform for Mad2:Mad2 dimerization at unattached kinetochores, which enables Mad2 to delay anaphase. Here, we show that mutations in Bub1 and within the Mad1 C-terminal domain impair the kinetochore localization of Mad1:Mad2 and abrogate checkpoint activity. Artificial kinetochore recruitment of Mad1 in these mutants co-recruits Mad2; however, the checkpoint remains non-functional. We identify specific mutations within the C-terminal head of Mad1 that impair checkpoint activity without affecting the kinetochore localization of Bub1, Mad1 or Mad2. Hence, Mad1 potentially in conjunction with Bub1 has a crucial role in checkpoint signalling in addition to presenting Mad2.

Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast).

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.

Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores.

The spindle assembly checkpoint (SAC) blocks entry into anaphase until all chromosomes have stably attached to the mitotic spindle through their kinetochores. The checkpoint signal originates from unattached kinetochores, where there is an enrichment of SAC proteins. Whether the enrichment of all SAC proteins is crucial for SAC signaling is unclear. Here, we provide evidence that, in fission yeast, recruitment of the kinase Mph1 is of vital importance for a stable SAC arrest. An Mph1 mutant that eliminates kinetochore enrichment abolishes SAC signaling, whereas forced recruitment of this mutant to kinetochores restores SAC signaling. In bub3Δ cells, the SAC is functional when only Mph1 and the Aurora kinase Ark1, but no other SAC proteins, are enriched at kinetochores. We analyzed the network of dependencies for SAC protein localization to kinetochores and identify a three-layered hierarchy with Ark1 and Mph1 on top, Bub1 and Bub3 in the middle, and Mad3 as well as the Mad1-Mad2 complex at the lower end of the hierarchy. If Mph1 is artificially recruited to kinetochores, Ark1 becomes dispensable for SAC activity. Our results highlight the crucial role of Mph1 at kinetochores and suggest that the Mad1-Mad2 complex does not necessarily need to be enriched at kinetochores for functional SAC signaling.

The spindle assembly checkpoint: progress and persistent puzzles.

The spindle assembly checkpoint is a conserved mitotic signalling pathway that ensures the equal segregation of chromosomes to daughter cells. Despite intensive work in many model organisms, key features of this safety mechanism remain unexplained. In the present review, I briefly summarize advances made in the last few years, and then focus on unexplored corners of this signalling pathway.

The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian.

Stochastic variation in gene products ("noise") is an inescapable by-product of gene expression. Noise must be minimized to allow for the reliable execution of cellular functions. However, noise cannot be suppressed beyond an intrinsic lower limit. For constitutively expressed genes, this limit is believed to be Poissonian, meaning that the variance in mRNA numbers cannot be lower than their mean. Here, we show that several cell division genes in fission yeast have mRNA variances significantly below this limit, which cannot be explained by the classical gene expression model for low-noise genes. Our analysis reveals that multiple steps in both transcription and mRNA degradation are essential to explain this sub-Poissonian variance. The sub-Poissonian regime differs qualitatively from previously characterized noise regimes, a hallmark being that cytoplasmic noise is reduced when the mRNA export rate increases. Our study re-defines the lower limit of eukaryotic gene expression noise and identifies molecular requirements for ultra-low noise which are expected to support essential cell functions.

The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian.

Gene expression inherently gives rise to stochastic variation ("noise") in the production of gene products. Minimizing noise is crucial for ensuring reliable cellular functions. However, noise cannot be suppressed below a certain intrinsic limit. For constitutively expressed genes, this limit is typically assumed to be Poissonian noise, wherein the variance in mRNA numbers is equal to their mean. Here, we demonstrate that several cell division genes in fission yeast exhibit mRNA variances significantly below this limit. The reduced variance can be explained by a gene expression model incorporating multiple transcription and mRNA degradation steps. Notably, in this sub-Poissonian regime, distinct from Poissonian or super-Poissonian regimes, cytoplasmic noise is effectively suppressed through a higher mRNA export rate. Our findings redefine the lower limit of eukaryotic gene expression noise and uncover molecular requirements for achieving ultralow noise, which is expected to be important for vital cellular functions.

Sock and Environmental Swabs as an Efficient, Non-Invasive Tool to Assess the Salmonella Status of Sow Farms.

Salmonellosis is the second most reported gastrointestinal infection in humans after campylobacteriosis and a common cause of foodborne outbreaks in the European Union (EU). In addition to consumption of contaminated animal-based foods, such as poultry, beef and eggs, pork is an important source of human salmonellosis outbreaks; therefore, Salmonella (S.) control should start in the early stages of pig production. To be able to implement effective control measures to reduce the risk of pigs being infected by Salmonella, it is important to identify the serovars circulating on farm within the different stages of production, including as early as sow and piglet breeding. The aim of the present study was to assess the Salmonella status of sow farms either producing their own finishers or delivering piglets to fattening farms with a known high serological prevalence identified within the QS Salmonella monitoring system. Overall, 97 (92.4%) of 105 investigated piglet-producing farms across Germany tested positive in at least one sample. Salmonella was detected in 38.2% of the sock and 27.1% of the environmental swab samples. S. Typhimurium was the most frequent serovar. In conclusion, sock and environmental swab samples are well suited for non-invasive Salmonella detection in different production units in farrowing farms. To establish a holistic Salmonella control program, all age classes of pig production should be sampled to enable intervention and implementation of countermeasures at an early stage if necessary.

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I.

Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.

Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin.

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono-orientation to bi-orientation.

Pomegranate: 2D segmentation and 3D reconstruction for fission yeast and other radially symmetric cells.

Three-dimensional (3D) segmentation of cells in microscopy images is crucial to accurately capture signals that extend across optical sections. Using brightfield images for segmentation has the advantage of being minimally phototoxic and leaving all other channels available for signals of interest. However, brightfield images only readily provide information for two-dimensional (2D) segmentation. In radially symmetric cells, such as fission yeast and many bacteria, this 2D segmentation can be computationally extruded into the third dimension. However, current methods typically make the simplifying assumption that cells are straight rods. Here, we report Pomegranate, a pipeline that performs the extrusion into 3D using spheres placed along the topological skeletons of the 2D-segmented regions. The diameter of these spheres adapts to the cell diameter at each position. Thus, Pomegranate accurately represents radially symmetric cells in 3D even if cell diameter varies and regardless of whether a cell is straight, bent or curved. We have tested Pomegranate on fission yeast and demonstrate its ability to 3D segment wild-type cells as well as classical size and shape mutants. The pipeline is available as a macro for the open-source image analysis software Fiji/ImageJ. 2D segmentations created within or outside Pomegranate can serve as input, thus making this a valuable extension to the image analysis portfolio already available for fission yeast and other radially symmetric cell types.

The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.