Biology subjects

Hildebrandt, F.

Publications and source records attributed to Hildebrandt, F..

11 recordsLinked to original sources

bioRxiv2025

NanTex enables computational multiplexing and phenotyping of organelles across super-resolution modalities

Super-resolution microscopy (SRM) enables nanoscale visualization of cellular organelles but remains constrained by spectral overlap, labeling requirements, and temporal offsets in multicolor imaging. We present NanTex, a deep learning framework that introduces nanotexture as a universal descriptor of subcellular organization and achieves probabilistic demixing of multiple organelles from single-channel SRM images. Unlike segmentation methods that enforce exclusivity, NanTex preserves overlapping morphologies, enabling faithful reconstruction even in crowded regions. Trained on small curated datasets with augmentation, NanTex generalized across SMLM, MINFLUX, STED, SIM, and live-cell Airyscan, with modality-specific retraining where necessary. Demonstrated on cytoskeletal, endomembrane, and metabolic organelles, NanTex achieved high-fidelity reconstructions and extended uniquely to live-cell imaging, where it eliminated temporal misalignment and enabled quantitative tracking of vesicle-like ER subdomains. Notably, NanTex enables quantitative dynamic readouts directly from single-channel live-cell data, revealing and tracking hidden subdomains in real time without the need for additional labels. Beyond multiplexing, NanTex supported computational phenotyping by distinguishing structurally distinct yet molecularly identical populations, exemplified by nocodazole-induced microtubule depolymerization and its glyoxal-mediated modulation. Together, these results establish nanotexture as a paradigm-shifting descriptor for organelle identity and position NanTex as a modality-agnostic, label-efficient, and live-cell-ready strategy for quantitative nanoscale biology. Significance StatementNanTex reframes multiplexing as texture-based demixing, introducing nanotexture as a universal fingerprint of organelle identity. By enabling quantitative, label-efficient reconstructions across SRM modalities and live-cell phenotyping, including vesicle tracking from single-channel data, NanTex opens a path to studying organelle remodeling and disease progression with unprecedented fidelity across SRM modalities.

biophysics↗

bioRxiv2025

A kidney specific mouse model to study the effects of in vivo induction of Yamanaka factors

IntroductionMaladaptive repair after acute kidney injury (AKI) leads to fibrosis and chronic kidney disease (CKD). Improving the resilience and stimulating tissue repair after injury is crucial to prevent AKI-to-CKD transition. Using a combination of transcription factors (Yamanaka factors Oct4, Klf4 and Sox2, "OKS") to partially reprogram tissues and enhance regeneration in vivo, could be a promising approach as shown by amelioration after various organ injury, yet not investigated for AKI to date. MethodsWe used a ubiquitously and kidney-specific transgenic mouse model to investigate OKS expression in kidney. In a kidney-specific model using Pax8-Cre, we then induced AKI via aristolochic acid (AA), simultaneously expressing OKS to determine potential protective effects after kidney injury. ResultsWe show that a ubiquitously expressing OKS-mouse model was not suitable due to toxic effects and limited kidney expression. In the Pax8-Cre mouse model, we observed expression almost exclusively to proximal tubules. While induction for more than 3 days caused dysplastic tumor formation, an induction regimen limited to 3 days was not able to improve phenotypic outcome after AA-injury. ConclusionPartial reprogramming of the kidney using OKS is feasible; however, it requires a delicate balance to the risk of oncogenic transformation. Determine a dose that effectively promotes repair without crossing the threshold into harmful effects remains a major challenge, posing significant safety concerns for translating the approach to humans in the near future.

developmental biology↗

bioRxiv2025

CAKUT variants in PRPF8, DYRK2, and CEP78: implications for splicing and ciliogenesis

IntroductionCongenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic kidney disease in children and young adults. Although over 50 monogenic causes have been identified, many remain unresolved. PRPF8 is a core spliceosome component, essential for pre-mRNA splicing, and further localizes to the distal mother centriole to promote ciliogenesis. MethodsWe performed trio exome sequencing in 208 CAKUT families and identified strong variants in PRPF8 and the EDD-DYRK2-DDB1VprBP complex. Functional validation included splicing assays in yeast (Saccharomyces cerevisiae), Sonic hedgehog (Shh) signaling in RPE-1 cells, co-immunoprecipitation for protein complex assembly, and in situ hybridization in mouse embryos. Protein interactions were modeled using AlphaFold. ResultsWe identified heterozygous de novo or inherited variants in PRPF8, DYRK2, DDB1, EDD and CEP78. Yeast assays revealed that while most PRPF8 variants preserved growth and splicing at consensus splice sites, the de novo PRPF8R1681W variant impaired splicing of non-consensus splice sites and was inviable at elevated temperature. CAKUT variants failed to rescue prp28-1 and U4-cs1 alleles but showed variant-specific synthetic interactions with brr2-1, including weak suppression or synthetic sickness at elevated temperatures. Shh signaling was reduced in [~]50% of PRPF8 variants expressed in RPE-1 cells. CEP78 truncating variants abrogated binding to CEP350 and VPRBP. Two DYRK2 variants disrupted EDD-DYRK2-DDB1VprBP complex formation without affecting kinase activity. In situ hybridization revealed strong Prpf8 expression in the developing collecting duct and urothelium. ConclusionVariants in PRPF8 and components of the EDD-DYRK2-DDB1VprBP complex may contribute to CAKUT through impaired pre-mRNA splicing and defective ciliogenesis. These findings uncover an entirely new functional network of candidate genes for CAKUT and ciliopathies, significantly broadening our understanding of disease mechanisms and offering novel entry points for mechanistic studies. Translational StatementOur study identifies a previously unrecognized molecular network involving PRPF8 and the EDD-DYRK2-DDB1VprBP complex, revealing a novel pathogenic mechanism in CAKUT. These results introduce a new class of candidate genes and pathways essential for kidney development. As the genetic etiology of CAKUT remains unknown in most patients, our findings underscore the need for targeted genetic testing and functional studies to enhance diagnosis, advance mechanistic insight, and enable more personalized clinical management.

genetics↗

bioRxiv2025

Mesoscale landscaping of the TRIM protein family reveals a novel human condensatopathy

The mesoscale organization of cells is central to cellular physiology and pathology. Cellular condensates often form via biomolecular phase separation, mediated by intrinsically disordered regions (IDRs) and represent a key mechanism for mesoscale organization. The TRI-partite Motif (TRIM) family of ubiquitin ligases is implicated in diverse cellular functions and disease, yet the role of biomolecular condensation in TRIM family organization remains understudied. Here, we systematically investigate the mesoscale localization of 72 TRIM proteins, revealing that a majority form condensates in distinct cellular compartments. IDR content correlates with dynamic condensate formation, suggesting a critical role in mesoscale organization. Focusing on TRIM8, associated with a neuro-renal disorder, we demonstrate that disease-causing truncations of the TRIM8 C-terminal IDR result in a condensatopathy, characterized by disrupted condensation, proteasomal regulation, and TAK1/NF{kappa}B signaling. Functional assays in cellular and animal models link these disruptions to podocyte dysfunction and impaired response to injury. Our findings establish a framework for understanding condensatopathies and the mesoscale principles governing TRIM family organization and function.

cell biology↗

bioRxiv2024

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders

All eukaryotes share a single-celled ancestor from [~]1.5-1.8 billion years ago, the Last Eukaryotic Common Ancestor (LECA). Roughly half of gene families found in modern eukaryotes were already present in LECA, forming molecular systems that continue to influence genetic diseases and traits today. To investigate these systems, we compared genes across 156 organisms to define a core set of protein-coding gene families likely present in LECA, with a quarter remaining uncharacterized. Integrating >26,000 mass spectrometry proteomics analyses from 31 species, we inferred higher-order complexes among these ancient proteins. This reconstructed interactome reveals both established and novel assemblies, offering a biochemical snapshot of LECAs organization. Finally, by exploring these ancient protein interactions, we found new human gene-disease associations for bone density and congenital birth defects, illustrating the value of ancestral protein networks for modern functional genetics.

evolutionary biology↗

bioRxiv2023

Host-Pathogen Interactions in the Plasmodium-Infected Mouse Liver at Spatial and Single-Cell Resolution

Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The spatial coordination of factors regulating immune responses and metabolic zonation during malaria infection, in the true tissue context, remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA-sequencing (snRNA-seq) over multiple time points during liver infection to delineate transcriptional programs of host-pathogen interactions across P. berghei-infected liver tissues. Our data suggest changes in gene expression related to lipid metabolism in response to Plasmodium infection in the proximity of infected hepatocytes, such as the modulation of the expression of genes involved in peroxisome proliferator-activated receptor pathway signaling. The data further indicate the presence of inflammatory hotspots with distinct cell type compositions and differential liver inflammation programs along the lobular axis in the malaria-infected tissues. Furthermore, a significant upregulation of genes involved in inflammation is observed in liver tissues of control mice injected with mosquito salivary gland components, which is considerably delayed compared to P. berghei infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection, and provides a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.

molecular biology↗

bioRxiv2023

scDual-Seq of Toxoplasma gondii-infected mouse bone marrow-derived dendritic cells reveals host cell heterogeneity and differential infection dynamics

Host-parasite interactions include complex interplays between an invading and a defending organism, each continuously adapting to gain the upper hand. The protozoan parasite, Toxoplasma gondii, can invade every nucleated cell type in a vertebrate host, including immune cells while the host mounts a protective response. Here, we utilize Dual-scSeq to parse out heterogeneous transcription of bone marrow-derived dendritic cells (BMDCs) infected with T. gondii type I, RH (LDM) or type II, ME49 (PTG) parasites, over multiple time points post infection. We find that the two parasite lineages distinctly manipulate two subpopulations of infected BMDCs. Co-expression networks establish host and parasite genes, with implications for modulation of host immunity and host-pathogen interactions. Integration of published data validates immune pathways and suggests novel candidate genes involved in host-pathogen interactions. This study aims to provide a comprehensive resource for future characterization of host-pathogen interplay among other protozoan parasites within their host niches, as well as that of bacterial and viral pathogens.

microbiology↗

bioRxiv2021

Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

cell biology↗

bioRxiv2020

CRISPR/Cas9 targeting Ttc30a mimics ciliary chondrodysplasia with polycystic kidney disease.

Skeletal ciliopathies (e.g. Jeune syndrome, short rib polydactyly syndrome, Sensenbrenner syndrome) are frequently associated with cystic kidney disease and other organ manifestations, but a common molecular mechanism has remained elusive. We established two models for skeletal ciliopathies (ift80 and ift172) in Xenopus tropicalis, which exhibited severe limb deformities, polydactyly, cystic kidneys, and ciliogenesis defects, closely matching the phenotype of affected patients. Employing data-mining and an in silico screen we identified candidate genes with similar molecular properties to genetically validated skeletal ciliopathy genes. Among four genes experimentally validated, CRISPR/Cas9 targeting of ttc30a replicated all aspects of the phenotypes observed in the models of genetically confirmed disease genes, including ciliary defects, limb deformations and cystic kidney disease. Our findings establish three new models for skeletal ciliopathies (ift80, ift172, ttc30a) and identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins implicating post-translational tubulin modifications in its pathogenesis.

developmental biology↗

bioRxiv2020

Cystin gene mutations cause autosomal recessive polycystic kidney disease associated with altered Myc expression

Mutation of the Cys1 gene underlies the renal cystic disease in the Cys1cpk/cpk (cpk) mouse that phenocopies human autosomal recessive polycystic kidney disease (ARPKD). Cystin, the protein product of Cys1, is expressed in the primary apical cilia of renal ductal epithelial cells. In previous studies, we showed that cystin regulates Myc expression via interaction with the tumor suppressor, necdin. Here, we demonstrate rescue of the cpk renal phenotype by kidney-specific expression of a cystin-GFP fusion protein encoded by a transgene integrated into the Rosa26 locus. In addition, we show that expression of the cystin-GFP fusion protein in collecting duct cells down-regulates expression of Myc in cpk kidneys. Finally, we report the first human patient with an ARPKD phenotype due to homozygosity for a predicted deleterious splicing defect in CYS1. These findings suggest that mutations in the Cys1 mouse and CYS1 human orthologues cause an ARPKD phenotype that is driven by overexpression of the Myc proto-oncogene. Translational StatementThe cystin-deficient cpk mouse is a model for the study of autosomal recessive polycystic kidney disease (ARPKD). We show that the cpk mouse phenotype is associated with altered Myc expression. To date, the clinical relevance of cystin deficiency to human disease was unclear, due to the absence of ARPKD cases associated with CYS1 mutations. We report the first case of ARPKD linked to a CYS1 mutation disrupting normal splicing. These findings confirm the relevance of cystin deficiency to human ARPKD, implicate Myc in disease initiation or progression, and validate the cpk mouse as a translationally relevant disease model.

molecular biology↗

bioRxiv2019

ADCK4 deficiency destabilizes the coenzyme Q complex, which is rescued by 2,4-dihydroxybenzoic acid treatment

ADCK4 mutations usually manifest as steroid-resistant nephrotic syndrome, and cause coenzyme Q10 (CoQ10) deficiency. However, the function of ADCK4 remains obscure. We investigated ADCK4 function using mouse and cell models. Podocyte-specific Adck4 deletion in mice significantly reduced survival and caused severe focal segmental glomerular sclerosis with extensive interstitial fibrosis and tubular atrophy, which were prevented by treatment with 2,4-dihydroxybenzoic acid (2,4-diHB), an analog of CoQ10 precursor molecule. ADCK4 knockout podocytes exhibited significantly decreased CoQ10 level, respiratory chain activity, mitochondrial potential, and dysmorphic mitochondria with loss of cristae formation, which were rescued by 2,4-diHB treatment, thus attributing these phenotypes to decreased CoQ10 levels. ADCK4 interacted with mitochondrial proteins including COQ5, and also cytoplasmic proteins including myosin and heat shock proteins. ADCK4 knockout decreased COQ complex levels, and the COQ5 level was rescued by ADCK4 overexpression in ADCK4 knockout podocytes. Overall, ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes.

genetics↗