Biology subjects

Hauf, S.

Publications and source records attributed to Hauf, S..

12 recordsLinked to original sources

bioRxiv2026

Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

Single-molecule RNA imaging using the MS2-MCP system has transformed the study of RNA biology across model organisms. However, this technology has remained unavailable for fission yeast (Schizosaccharomyces pombe), even though fission yeast is a central model for eukaryotic gene expression. Achieving single-molecule sensitivity requires identifying a narrow optimum where RNA labels are sufficiently bright while background fluorescence remains minimal. We have now accomplished this for S. pombe by systematically optimizing MCP expression and localization--screening a panel of constitutive S. pombe promoters and evaluating combinations of nuclear localization and export signals (NLSs and NESs). The resulting, successful constructs use tandem StayGold as the MCP fluorescent tag, taking advantage of its superior photostability. Together with optimized vectors for MS2 stem-loop tagging of endogenous transcripts, these tools enable single-molecule RNA imaging in fission yeast, opening the door to quantitative analyses of RNA dynamics in this core genetic model. Summary statementWe establish single-molecule RNA visualization with MS2/MCP for fission yeast, S. pombe, an important model organism to study RNA biology.

molecular biology↗

bioRxiv2026

Chromosome segregation synchrony in S. pombe is noise-limited and arises without positive feedback

Anaphase is a key cell cycle transition that ensures faithful genome inheritance. At anaphase onset, sister chromatids separate abruptly and synchronously upon activation of the protease separase. Major cell cycle transitions often involve positive feedback, which contributes to their abruptness and irreversibility; however, whether such feedback is required for anaphase remains unclear. Here, we analyze sister chromatid separation dynamics in fission yeast using high-resolution live-cell imaging and computational modeling. We find that anaphase synchrony relies on fast degradation of the separase inhibitor securin, but does not require separase-mediated positive feedback. Hence, sister chromatid separation, being inherently irreversible, may be one of the few major cell cycle transitions that can proceed without positive feedback. A stochastic model fitted to the data revealed that separation synchrony is limited by stochasticity resulting from small-number effects. Together, these results support a feedback-independent mechanism for anaphase onset and identify molecular noise as a fundamental constraint on its temporal precision.

cell biology↗

bioRxiv2026

Love-thy-neighbor: Neural networks for tracking and lineage tracing in budding yeast

Tracking and lineage tracing are widely needed tasks in biological image analysis. For cells that grow and divide, tracking is challenging because cells change in number, shape, and size throughout a recording. As the time interval between images increases, it becomes more difficult to establish correspondences between cells across timepoints. Consequently, tracking has to be performed between consecutive or temporally close images, which leads to exponentially decreasing tracking accuracy and thus high sensitivity to error rates. For budding yeast, this challenge is further heightened by the similarity of cells in colonies, their dense packing, the asymmetric nature of cell divisions, and movement due to growth of the colony. A related task, lineage tracing, is similarly challenging without fluorescent markers due to multiple potential mother cells surrounding a new daughter cell. Here, we present neural networks for budding yeast tracking and lineage tracing, named LYN-track and LYN-trace, respectively. These methods leverage fine geometric features of cells and their neighborhoods. To train and test the algorithms, we recorded and annotated new budding and fission yeast microscopy movies (78,852 frame-to-frame tracklets, 2,512 images), which we make freely available. On these and existing datasets, our neural network-based methods demonstrate robust, above state-of-the-art performance. Both tools have been integrated into graphical user interfaces (GUIs), available on Github, and can be straightforwardly retrained with custom data if desired.

bioinformatics↗

bioRxiv2025

A distinct phase of cyclin B (Cdc13) nuclear export at mitotic entry in S. pombe

In eukaryotes, cell division requires coordination between the nucleus and cytoplasm. Entry into cell division is driven by cyclin-dependent kinases (CDKs), which need a cyclin binding partner for their activity. In Schizosaccharomyces pombe (fission yeast), the B-type cyclin Cdc13 is essential and sufficient for cell cycle progression and is strongly enriched in the nucleus. Here, we show that a fraction of Cdc13 is exported from the nucleus to the cytoplasm just prior to mitosis. This export could be critical to propagate CDK activity throughout the cell. Mutating three Cdc13 nuclear localization signals (NLSs) led to precocious enrichment of Cdc13 in the cytoplasm but did not accelerate mitotic entry, indicating that the export is not sufficient to trigger entry into mitosis. The export coincides with spindle pole body integration into the nuclear envelope and may be required to coordinate nuclear and cytoplasmic signaling required for this integration. The onset and stop of Cdc13 nuclear export are remarkably abrupt, underscoring that S. pombe mitotic entry consists of several switch-like transitions over the course of minutes. Our findings add another instance to the various cyclin nuclear transport events known to occur at critical cell cycle transitions throughout eukaryotes.

cell biology↗

bioRxiv2025

RNA condensates as platforms for prebiotic chemistry

The RNA world hypothesis posits the existence of life-like assemblies that consisted mostly of RNA. However, questions remain regarding the emergence of RNA catalysis, stability, reactant availability, and compartmentalization of genetic material. At acidic pH, short RNAs (average {approx} 20 nt) readily phase-separate into a condensed phase en-riched with long RNA. These RNA condensates stably compartmentalize RNA as well as DNA and maintain stable identities even in the absence of membranes. Additionally, the RNA condensates concentrate ions, small molecules, phospholipids, peptides, ri-bozymes, and proteins. Beyond enriching such diverse components, RNA condensates function as microreactors with two catalytic capabilities: they physically enhance reac-tion rates by concentrating reactants within a confined space and simultaneously act as inherent catalysts that directly facilitate chemical transformations. RNA condensates can also support ribozyme and enzymatic activity. Together, these findings suggest that RNA phase separation may have played a crucial role in lifes origins by providing compartmentalization, inherent catalytic activity, and molecular enrichment of long, potentially catalytic biopolymers.

biochemistry↗

bioRxiv2023

Rapid enzymatic detection of Shigatoxin-producing E. coli using fluorescence-labeled oligonucleotide substrates

Shigatoxin-producing E. coli (STEC) are important human pathogens causing disease ranging from diarrhea to severe hemolytic uremic syndrome. As STEC are transmitted via animals, food, and water, and may produce large outbreaks, their timely and qualified detection including isolate recovery is of high importance, but challenging and labor-intense. Thus, the availability of an easy-to-perform rapid test would be a tremendous advance. Since the common feature and major virulence factor of otherwise multifaceted STEC is the Shiga toxin (Stx), we developed a detection method for Stx, specifically for its catalytic RNA-N-glycosidase activity targeting the Sarcin Ricin Loop (SRL) of 28S ribosomal RNA. To this end, synthetic ssDNA substrates mimicking the SRL were designed and linked to a fluorophore and quencher pair, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 minutes incubation. Importantly, different Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. In conclusion, the assay offers rapid and facile detection of STEC based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak and source detection, and simplify research for antimicrobials.

microbiology↗

bioRxiv2023

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.

molecular biology↗

bioRxiv2021

Speed fluctuations of bacterial replisomes

Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly five-fold between 17{degrees}C and 37{degrees}C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate. We interpret this correlation as a speed-error trade-off in DNA replication. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

biophysics↗

bioRxiv2021

Mitotic checkpoint gene expression is tuned by coding sequences

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Proper functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about SAC gene expression. Here, we show in fission yeast (S. 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 supported by a high frequency of non-optimal codons. In contrast, mad1+ mRNA has a short half-life despite a low frequency of non-optimal codons and despite the lack of known 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 for 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.

cell biology↗

bioRxiv2021

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 channel substrates to the proteasome. 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, this establishes Cdc48 as a key regulator of 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.

cell biology↗

bioRxiv2020

Pomegranate: a segmentation and 3D reconstruction pipeline for fission yeast

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 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.

cell biology↗

bioRxiv2019

Budding yeast centromeric DNA and A+T rich bacterial DNA can function as centromeres in the fission yeast Schizosaccharomyces pombe.

Eukaryotic centromeric DNA is famously variable in evolution but currently, this cannot be reconciled with the conservation of eukaryotic centromere function. It seems likely that centromeric DNA from different organisms contains conserved functionally important features but the identity of these features is unknown. The point centromeres of the budding yeast Saccharomyces cerevisiae and the regional centromeres of the fission yeast Schizosaccharomyces pombe are separated by 350 million years of evolution and are canonical examples of the paradoxical relationship1 between centromeric DNA sequence and function. We have established a centromere-replacement strategy in Schizosaccharomyces pombe in order to resolve this paradox experimentally. Centromere-replacement shows that an A+T rich bacterial DNA sequence has weak centromere function and that elements of the Saccharomyces cerevisiae centromere embedded in short sequences from the non-centromeric S. pombe wee1 gene function almost as well as native S. pombe centromeric DNA. These observations demonstrate that determinants of centromere function are held in common by the budding and fission yeasts and that A+T rich DNA is both necessary and sufficient for function in S. pombe. Given the evolutionary distance between these yeasts, it is likely that A+T rich DNA has centromere function in a wide variety of eukaryotes. Centromere-replacement uses unidirectional serine recombinases that work well in many organisms2 3 and our experimental strategy should allow this idea to be tested in other eukaryotes.

genetics↗