Biology subjects

Stadager, J.

Publications and source records attributed to Stadager, J..

3 recordsLinked to original sources

bioRxiv2026

Interplay between β-catenin transcriptional and cell-cell junction activity regulates homeostasis and collective dynamics in the intestinal epithelium

Wnt signaling is crucial for stem cell maintenance during development and homeostasis, and its dysregulation is implicated in cancers across multiple tissues. {beta}-catenin, the primary mediator of canonical Wnt signaling, is also a crucial adaptor protein at cell-cell junctions and is required for maintaining tissue integrity. How the interplay and potential competition between these discrete functions of {beta}-catenin influences tissue homeostasis and organization is not well understood. Here, we address this open question in the intestinal epithelium, which is highly dependent on Wnt signaling during homeostasis and regeneration. Ectopic activation of Wnt signaling in intestinal organoids by pharmacological or genetic perturbation leads to tissue fluidification and loss of epithelial integrity, while disrupting Wnt activity perturbs intracellular {beta}-catenin localization and dynamics at cell-cell junctions. Stabilization of cell-cell junctions increases {beta}-catenin accumulation at junctions at the expense of nuclear localization, while junctional perturbation leads to {beta}-catenin release from junctions into the cytoplasm, leading to dysregulation of Wnt signaling. Together, our findings indicate that in tissues with active Wnt signaling, the interplay between junction- and signaling-associated {beta}-catenin is crucial for stem cell regulation and tissue homeostasis.

cell biology↗

bioRxiv2024

CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. MotivationThe CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing1-8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS)9-12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.

biochemistry↗

bioRxiv2021

Cell-to-cell heterogeneities during extrinsic apoptosis arise from cell cycle progression and transmitotic apoptosis resistance

Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating biologics. Here, we monitored cell cycle progression at minutes resolution to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour for mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation from mid S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 by BH3-mimetics suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival from apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival from inefficient apoptosis induction. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis signalling.

cell biology↗