Biology subjects

Tholen, S.

Publications and source records attributed to Tholen, S..

9 recordsLinked to original sources

bioRxiv2026

Distinct effects of hypomorphic IFT and dynein-2 skeletal ciliopathy disease alleles on chondrogenic differentiation, ECM composition and wnt signalling in ATDC5 derived cartilage-like organoids

Dysfunction of ciliary intraflagellar transport (IFT) and dynein-2 genes causes severe developmental defects, including skeletal phenotypes characterized by shortened ribs and long bones and polydactyly. Specific gene-phenotype associations suggest individual functions of the different IFT/dynein-2 proteins in development. Since null models disrupt ciliogenesis and hence are not suitable to study individual protein functions, we recreated human hypomorphic disease alleles in IFT-A (IFT43 p.M1V), IFT-B (IFT74 deletion of exon 2), and dynein-2 (WDR60 p.A911V), alongside a WDR60 null model in ATDC5 chondrocyte precursor cells. Hypomorphic mutants did not show alterations in ciliation efficiency or cilia length but displayed distinct defects in IFT88 localization, indicating impaired intraflagellar transport. Despite altered IFT, Hedgehog signalling responses were variably affected across the different genotypes. Transcriptomic analysis revealed concurrent increases in canonical Wnt signalling and expression of genes related to late skeletal development in WDR60 A911V and IFT74 del ex 2 mutants, but not in IFT43 M1V or WDR60 loss-of-function mutants. These changes were accompanied by alterations in ECM composition. In addition, all hypomorphic mutants showed reduced levels of the non-canonical Wnt ligand WNT5A in ECM proteomic analyses. Interestingly, loss of cilia in WDR60 loss-of-function mutants had only modest effects on chondrogenic differentiation and ECM composition. Overall, our data provide new evidence of genotype-dependent altered ECM composition as well as dysregulation of canonical and non-canonical Wnt signalling and accelerated chondrocyte differentiation in skeletal ciliopathies. Furthermore, our findings suggest a modulatory rather than essential role of the primary cilium for ATDC5 cell differentiation.

cell biology↗

bioRxiv2026

Deep phenotyping of ATDC5-derived in vitro cartilage organoids

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.

cell biology↗

bioRxiv2025

Vertical RAS-pathway inhibition in pancreatic cancer drives therapeutically exploitable mitochondrial alterations

Background & AimsOncogenic KRAS mutations drive metabolic rewiring in pancreatic ductal adenocarcinoma (PDAC). Src-homology 2 domain-containing phosphatase 2 (SHP2) is essential for full KRAS activity and promising dual SHP2/mitogen-activated protein kinase (MAPK) inhibition is currently being tested in clinical trials. Exploitable metabolic adaptations may contribute to an invariably evolving resistance. MethodsTo understand the metabolic changes induced by dual inhibition, we comprehensively tested cell lines, endogenous tumor models, and patient-derived organoids representing the full spectrum of PDAC molecular subtypes. ResultsWe find that dual SHP2/mitogen-activated protein kinase kinase (MEK1/2) inhibition induces major mitochondrial alterations, elevates reactive oxygen species (ROS) levels and triggers a lipid peroxidase dependency. While anabolic pathways, glycolysis and autophagy were also affected, mitochondrial alterations persisted longterm into a therapy resistant state. ConclusionsThe resulting vulnerability to induction of ferroptotic cell death via combined SHP2/MEK1/2 and glutathione peroxidase (GPX4) inhibition provides a metabolic lever to reinforce RAS-pathway inhibition for targeted PDAC treatment.

cancer biology↗

bioRxiv2024

The RRM domain-containing protein Rbp3 interacts with ribosomes and the 3' ends of mRNAs encoding photosynthesis proteins

RNA recognition motif (RRM) domain proteins are crucial RNA-binding proteins (RBPs) across all domains of life. In cyanobacteria, single RRM domain proteins are involved in mRNA targeting to the thylakoid membrane and acclimation to certain stress conditions, but many details of their physiological functions and molecular targets have remained unknown. The model cyanobacterium Synechocystis sp. PCC 6803 has a family of three genes encoding the RRM domain-containing proteins Rbp1, Rbp2 and Rbp3. Here, we verified the RNA-binding activity of Rbp3 in vivo and show that cells of a {Delta}rbp3 deletion strain had a lower PSI:PSII ratio and decreased pigment content and were significantly smaller than wild-type cells. To identify the set of interacting molecules, co-immunoprecipitation experiments were performed with a strain expressing a C-terminally FLAG-tagged Rbp3. Mass spectrometry of the elution fraction suggested physical proximity between Rbp3, ribosomes, and a very small number of other proteins. The most highly enriched transcript in the co-eluting RNA fraction was the psaAB mRNA. This was corroborated by fluorescent in situ hybridization (FISH) analyses showing decreased psaA mRNA signals in {Delta}rbp3, and colocalization with Rbp3-GFP in the wild type. Other mRNAs co-enriched with Rbp3 encode thylakoid, plasma membrane and carboxysome proteins. Binding assays using Bio-layer Interferometry validated the Rbp3-psaAB mRNA interaction, indicating a preference for folded RNA segments near or overlapping the respective stop codons. Significance statementThe mechanisms by which proteins are produced at specific sites and inserted into the intricate membrane systems of photosynthetic cyanobacteria are only partially understood. While RRM domain proteins are well-studied RNA-binding proteins in eukaryotes, their functions in bacteria remain underexplored. This study reveals that the RRM domain protein Rbp3 in the cyanobacterium Synechocystis sp. PCC 6803 binds mRNAs encoding photosynthetic proteins, plasma membrane proteins and carboxysome proteins and localizes near ribosomes. The bound RNA segments are typically near the ends of coding regions, or in 5' untranslated regions. These findings suggest that Rbp3 is involved in targeting mRNAs to various intracellular locations by interacting with structural elements within these mRNA molecules.

microbiology↗

bioRxiv2024

The Role of the Co-Chaperone DNAJB11 in Polycystic Kidney Disease: Molecular Mechanisms and Cellular Origin of Cyst Formation

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), which are required for the regulation of the renal tubular diameter. Loss of polycystin function results in cyst formation. Atypical forms of ADPKD are caused by mutations in genes encoding endoplasmic reticulum (ER)-resident proteins through mechanisms that are not well understood. Here, we investigate the function of DNAJB11, an ER co-chaperone associated with atypical ADPKD. We generated mouse models with constitutive and conditional Dnajb11 inactivation and Dnajb11-deficient renal epithelial cells to investigate the mechanism underlying autosomal dominant inheritance, the specific cell types driving cyst formation, and molecular mechanisms underlying DNAJB11-dependent polycystic kidney disease. We show that biallelic loss of Dnajb11 causes cystic kidney disease and fibrosis, mirroring human disease characteristics. In contrast to classical ADPKD, cysts predominantly originate from proximal tubules. Cyst formation begins in utero and the timing of Dnajb11 inactivation strongly influences disease severity. Furthermore, we identify impaired PC1 cleavage as a potential mechanism underlying DNAJB11-dependent cyst formation. Proteomic analysis of Dnajb11- and Pkd1-deficient cells reveals common and distinct pathways and dysregulated proteins, providing a foundation to better understand phenotypic differences between different forms of ADPKD.

molecular biology↗

bioRxiv2024

Characterization of YlxR/Ssr1238, a conserved RNA binding protein in a model cyanobacterium

Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Structural prediction suggests an RNA-binding interface for the protein YlxR/Ssr1238 in the cyanobacterium Synechocystis 6803. Two pairs of cysteine residues are arranged as possibly coordinating an Fe-S cluster and appear widely conserved in the homologous proteins of other cyanobacteria. Overexpression of Ssr1238 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript derived from gene ssl3177, which encodes a central enzyme in cell wall remodeling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and participates in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the B. subtilis YlxR protein functions as an RNase P modulator (RnpM), but suggest additional functionalities and extend its proposed role to the phylum cyanobacteria.

microbiology↗

bioRxiv2020

Flattened circadian glucocorticoid oscillations cause obesity due to increased lipid turnover and lipid uptake

Chronic stressors flatten diurnal glucocorticoid oscillations, resulting in obesity, insulin resistance, and diabetes. How such flattened diurnal glucocorticoid oscillations increase fat storage is unknown. Here we investigated the consequences in mice and found that flattening of glucocorticoid oscillations results not only in body weight gain, mainly due to increases in white fat depot mass, but also leads to hyperinsulinemia and fat accumulation in brown adipose tissue. A transcriptomic analysis of white and brown adipose tissues revealed that flattened glucocorticoid oscillations cause dysregulated lipid metabolism with a prominent role of the fatty acid transporter Cd36 and insulin-driven adipocyte hypertrophy. Indeed, Cd36 knockout mice are partially protected against the adverse effects of flattened GC oscillations including body weight gain and lipid accumulation in the brown and visceral white fat depots. These results show the molecular mechanisms how flattened glucocorticoid oscillations can cause obesity and diabetes.

physiology↗

bioRxiv2020

The highly expressed lipid buffer FABP4 enforces adipocyte cell identity by driving the initial cell differentiation process

How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given that critical transcription factors are often not unique to a differentiation process and the fluctuating signaling environment in which cells exist. Here we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after upregulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of adipogenesis, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty-acid loaded FABP4 binds to and increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state. HIGHLIGHTSO_LIFatty-acid loaded FABP4 binds to and increases PPARG expression, thereby turning on PPARG positive feedback loops that further increase PPARG expression. C_LIO_LIFABP4 critically controls the second phase of adipogenesis between activation of the feedback loops and reaching the threshold to differentiate. C_LIO_LIOnly a small fraction ([~]10%) of the FABP4 levels typically attained in mature fat cells is needed to commit cells to the differentiated state, thus providing an explanation for why maintenance of the mature adipocyte state is so robust. C_LI

cell biology↗

bioRxiv2018

Transcription factor dynamics reveals a circadian code for fat cell differentiation

Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern during stress and disease correlates with increased fat mass and obesity in humans, raising the intriguing question of how hormone secretion dynamics affect the process of adipocyte differentiation. By using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system, leading to very low adipocyte differentiation rates. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushings disease, and other conditions for which glucocorticoid secretion loses its pulsatility can lead to obesity. Given the ubiquitous nature of oscillating hormone secretion in mammals, the filtering mechanism we uncovered may represent a general temporal control principle for differentiation.\n\nHIGHLIGHTO_LIWe found that the fraction of differentiated cells is controlled by rhythmic and pulsatile hormone stimulus patterns.\nC_LIO_LITwelve hours is the cutoff point for daily hormone pulse durations below which cells fail to differentiate, arguing for a circadian code for hormone-induced cell differentiation.\nC_LIO_LIIn addition to fast positive feedback such as between PPARG and CEBPA, the adipogenic transcriptional architecture requires added parallel slow positive feedback to mediate temporal filtering of circadian oscillatory inputs\nC_LI

cell biology↗