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.

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.

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.

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.

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.

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.

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.

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.

Construction, Growth, and Harvesting of Fission Yeast Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Strains.

Stable isotope labeling by amino acids in cell culture (SILAC) enables the relative quantification of protein amounts and posttranslational modifications in complex biological samples through the use of stable heavy isotope-labeled amino acids. Here we describe methods for the application of SILAC to fission yeast Schizosaccharomyces pombe using either labeled lysine or a combination of labeled lysine and labeled arginine. The latter approach is more complicated than the use of labeled lysine alone but may yield a more comprehensive (phospho)proteomic analysis. The protocol includes methods for construction of SILAC-compatible strains, growth of cultures in labeled medium, cell harvesting, and protein extraction.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast.

Modern mass spectrometry (MS)-based approaches are capable of identifying and quantifying thousands of proteins and phosphorylation events in a single biological experiment. Here we present a (phospho)proteomic workflow based on in-solution proteome digestion of samples labeled by stable isotope labeling by amino acids in cell culture (SILAC) and phosphopeptide enrichment using strong cation exchange (SCX) and TiO2 chromatographies. These procedures are followed by high-accuracy MS measurement on an Orbitrap mass spectrometer and subsequent bioinformatic processing using MaxQuant software.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast.

Shotgun proteomics combined with stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach to quantify proteins and posttranslational modifications across the entire proteome. SILAC technology in Schizosaccharomyces pombe must cope with the "arginine conversion problem," in which isotope-labeled arginine is converted to other amino acids. This can be circumvented by either using stable isotope-marked lysine only (as opposed to the more standard lysine/arginine double labeling) or using yeast genetics to create strains that only very inefficiently convert arginine. Both strategies have been used successfully in large-scale (phospho)proteomics projects in S. pombe Here we introduce methods for performing a typical SILAC-based experiment in fission yeast, including generation of SILAC-compatible strains, sample preparation, and measurement by mass spectrometry.

Different Functionality of Cdc20 Binding Sites within the Mitotic Checkpoint Complex.

The mitotic checkpoint is a cellular safeguard that prevents chromosome missegregation in eukaryotic cells [1, 2]. Suboptimal functioning may foster chromosome missegregation in cancer cells [3]. Checkpoint signaling produces the "mitotic checkpoint complex" (MCC), which prevents anaphase by targeting Cdc20, the activator of the anaphase-promoting complex/cyclosome (APC/C). Recent biochemical and structural studies revealed that the human MCC binds two Cdc20 molecules, one (Cdc20M) through well-characterized, cooperative binding to Mad2 and Mad3/BubR1 (forming the "core MCC") and the other one (Cdc20A) through additional binding sequences in Mad3/BubR1 [4-6]. Here, we dissect the different functionality of these sites in vivo. We show in fission yeast that, at low Cdc20 concentrations, Cdc20M binding is sufficient for checkpoint activity and Cdc20A binding becomes dispensable. Cdc20A binding is mediated by the conserved Mad3 ABBA-KEN2-ABBA motif [7, 8], which we find additionally required for binding of the MCC to the APC/C and for MCC disassembly. Strikingly, deletion of the APC/C subunit Apc15 mimics mutations in this motif, revealing a shared function. This function of Apc15 may be masked in human cells by independent mediators of MCC-APC/C binding. Our data provide important in vivo support for the recent structure-based models and functionally dissect three elements of Cdc20 inhibition: (1) sequestration of Cdc20 in the core MCC, sufficient at low Cdc20 concentrations; (2) inhibition of a second Cdc20 through the Mad3 C terminus, independent of Mad2 binding to this Cdc20 molecule; and (3) occupancy of the APC/C with full MCC, where Mad3 and Apc15 are involved.

Micromanaging checkpoint proteins.

The kinase Mps1, long known to be the 'boss' in mitotic checkpoint signaling, phosphorylates multiple proteins in the checkpoint signaling cascade.

Time To Split Up: Dynamics of Chromosome Separation.

The separation of chromosomes in anaphase is a precarious step in the cell cycle. The separation is irreversible, and any error can lead to cell death or genetic instability. Chromosome separation is controlled by the protease separase. Here we discuss recent work that has revealed additional layers of separase regulation and has deepened our understanding of how separase activation is coordinated with other events of mitotic exit.

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.

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.

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.

Slow checkpoint activation kinetics as a safety device in anaphase.

Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1, 2] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3, 4]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5, 6]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7-12] and to the inactivation of the checkpoint by the time of anaphase [10, 11, 13, 14]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions.