Biology subjects

Cuylen-Haering, S.

Publications and source records attributed to Cuylen-Haering, S..

9 recordsLinked to original sources

bioRxiv2026

In-cell structures visualize human pre-ribosome assembly in the nucleolus

Ribosome biogenesis is an essential multi-step process. In eukaryotes, it is orchestrated within the nucleolus, a hallmark multi-layered compartment of the nucleus. Structures of pre-ribosomes have been characterized in vitro, but their assembly pathways within human nucleoli have not been described. Here, we used cryogenic correlative light and electron tomography to visualize the molecular landscapes within HeLa cell nucleoli and obtained in-cell structural snapshots of both ribosomal subunit precursors, the SSU processome and the pre-60S. These recapitulate major states resolved previously in vitro while additionally revealing critical interaction partners, including the RNA exosome, rixosome, and nuclear export receptor CRM1-RanGTP. We further show how pre-ribosome assembly is altered upon RNA polymerase I inhibition. Our study combines molecular structures with cellular context to elucidate the spatiotemporal assembly pathways and regulation of human ribosome biogenesis.

cell biology↗

bioRxiv2025

Ki-67 shapes the nucleolus by anchoring chromatin via its amphiphilic properties

The nucleolus, a membrane-less organelle essential for ribosome biogenesis, adopts variable shapes across cell types and in response to environmental conditions, yet the mechanisms regulating its morphology and functional implications remain unclear. Using a high-throughput screen, we identify the proliferation marker Ki-67 as a central regulator of nucleolar shape. Ki-67 localises to the chromatinnucleolus interface, where its depletion induces nucleolar rounding and reduces chromatin enrichment within and around the nucleolus. This effect is driven by amphiphilic properties conferred by two distinct affinity domains separated by a spacer. Given that chromatin loss is a common feature of rounded nucleoli in our screen, and acute chromatin digestion also induces rounding, we propose that the chromatin environment in and around the nucleolus plays a key role in determining nucleolar shape. Our study elucidates a novel Ki-67-mediated chromatin anchoring mechanism, tightly linking nucleolar shape to genome organisation and expanding our understanding of condensate morphology.

cell biology↗

bioRxiv2024

Charge distribution of coating brush drives inter-chromosome attraction

The condensation of charged polymers is an important driver for the formation of biomolecular condensates. Recent experiments suggest that this mechanism also controls the clustering of eukaryotic chromosomes during the late stages of cell division. In this process, inter-chromosome attraction is driven by the condensation of cytoplasmic RNA and Ki-67, a charged intrinsically disordered protein that coats the chromosomes as a brush. Attraction between chromosomes has been shown to be specifically promoted by a localized charged patch on Ki-67, although the physical mechanism remains unclear. To elucidate this process, we combine here coarse-grained simulations and analytical theory to study the RNA-mediated interaction between charged polymer brushes on the chromosome surfaces. We show that the charged patch on Ki-67 leads to inter-chromosome attraction via RNA bridging between the two brushes, whereby the RNA preferentially interacts with the charged patches, leading to stable, long-range forces. By contrast, if the brush is uniformly charged, bridging is basically absent due to complete adsorption of RNA onto the brush. Moreover, the RNA dynamics becomes caged in presence of the charged patch, while remaining diffusive with uniform charge. Our work sheds light on the physical origin of chromosome clustering, while also suggesting a general mechanism for cells to tune work production by biomolecular condensates via different charge distributions.

biophysics↗

bioRxiv2024

A multivalent adaptor mechanism drives the nuclear import of proteasomes

Nuclear protein homeostasis, including the turnover of transcription factors, critically depends on nuclear proteasomes. After each cell division, proteasomes need to be re-imported into the newly formed nucleus in a highly dynamic process that requires the largely unstructured protein AKIRIN2. However, how AKIRIN2 orchestrates this process and, more generally, how large protein complexes are translocated into the nucleus remains poorly understood. Here, we have used an integrated approach combining protein-wide saturation mutagenesis screens, cryoEM, and biochemical reconstitution to characterize AKIRIN2 as a scaffold protein that coordinates the stepwise assembly of an importin cluster around the proteasome. Through surveying every possible single amino acid substitution in AKIRIN2 using FACS- and microscopy-based genetic screens, we establish a comprehensive map of functionally relevant residues and binding interfaces in structured and disordered protein regions. Integrating these results with cryoEM analysis reveals a wing helix in a disordered region of AKIRIN2 that plays a crucial role in stabilizing proteasome interactions. Upon primary binding, AKIRIN2 homodimers recruit the importin IPO9, which in turn facilitates the binding of a second AKIRIN2 homodimer that recruits additional importins. Together, this multivalent molecular assembly amplifies the number of nuclear localisation signals and, thereby, triggers efficient proteasome translocation into the nucleus. Inside the nucleus, RanGTP rapidly dissociates importins, and AKIRIN2 is degraded by the proteasome in a ubiquitin-independent manner. Beyond mechanistically resolving the nuclear import of proteasomes, we propose that multivalent adaptor proteins like AKIRIN2 orchestrate the import of other macromolecular complexes and thereby dynamically control the composition of the nuclear proteome.

molecular biology↗

bioRxiv2023

A liquid-like coat mediates chromosome clustering during mitotic exit

SummaryThe individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces driven by phase separation promote clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate the mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.

cell biology↗

bioRxiv2022

Genetically encoded multimeric tags for intracellular protein localisation in cryo-EM

Cryo-electron tomography is a powerful label-free tool for visualizing biomolecules in their native cellular context at molecular resolution. However, the precise localisation of biomolecules of interest in the tomographic volumes is challenging. Here, we present a tagging strategy for intracellular protein localisation based on genetically encoded multimeric particles (GEMs). We show the applicability of drug-controlled GEM labelling of endogenous proteins in cryo-electron tomography and cryo-correlative fluorescence imaging in human cells.

cell biology↗

bioRxiv2021

Unsupervised temporal consistency improvement for microscopy video segmentation with Siamese networks

We introduce a simple mechanism by which a CNN trained to perform semantic segmentation of individual images can be re-trained - with no additional annotations - to improve its performance for segmentation of videos. We put the segmentation CNN in a Siamese setup with shared weights and train both for segmentation accuracy on annotated images and for segmentation similarity on unlabelled consecutive video frames. Our main application is live microscopy imaging of membrane-less organelles where the fluorescent groundtruth for virtual staining can only be acquired for individual frames. The method is directly applicable to other microscopy modalities, as we demonstrate by experiments on the Cell Segmentation Benchmark. Our code is available at https://github.com/kreshuklab/learning-temporal-consistency.

bioinformatics↗

bioRxiv2020

Mapping of the nucleolar proteome reveals spatiotemporal organization related to intrinsic protein disorder

The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment (nucleoli rim). We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

cell biology↗

bioRxiv2018

Condensin controls cellular RNA levels through the accurate segregation of chromosomes instead of directly regulating transcription

Condensins are genome organisers that shape chromosomes and promote their accurate transmission. Several studies have also implicated condensins in gene expression, although the mechanisms have remained enigmatic. Here, we report on the role of condensin in gene expression in fission and budding yeasts. In contrast to previous studies, we provide compelling evidence that condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis. We further show that the changes in gene expression in post-mitotic fission yeast cells that result from condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite condensin inactivation. Thus, chromosome instability, rather than a direct role of condensin in the transcription process, changes gene expression. This knowledge challenges the concept of gene regulation by canonical condensin complexes.

molecular biology↗