Biology subjects

Trcka, D.

Publications and source records attributed to Trcka, D..

6 recordsLinked to original sources

bioRxiv2026

Spatiotemporal remodeling of cytoskeletal and junction networks during somatic cell reprogramming

Summary/AbstractReprogramming somatic cells into induced pluripotent stem cells involves a dramatic reorganization of the cytoskeleton and junctions during the critical mesenchymal-to-epithelial transition stage. While protein abundance changes have been profiled, the spatiotemporal dynamics of protein-protein associations involving these structural components remain poorly resolved. Here, we present a time-resolved proximity proteomics resource that maps cytoskeletal and junctional remodeling across 27 baits during the early stages of reprogramming. We identified over 1100 high-confidence interactions, including many not previously reported, capturing the dynamic reorganization of cell architecture. By integrating proximity-dependent biotinylation with quantitative proteomics, we distinguished spatial relocalizations from abundance-driven effects. Dynamic redistributions of actin regulators and desmosomal proteins were observed, and a targeted short interfering RNA screen uncovered early acting structural proteins essential for colony formation. Our findings reveal adhesion and cytoskeletal maturation as structural bottlenecks in reprogramming and provide a broadly applicable framework for mapping subcellular remodeling during dynamic cell fate transitions.

systems biology↗

bioRxiv2024

LINE1 promotes nuclear compartmentalization to repress the 8-cell state in embryonic stem cells

The family of LINE1 transposable elements underwent a massive expansion in mammalian genomes. While traditionally viewed as a mutagenic selfish element, recent studies point to roles for LINE1 in early mouse development, T cell quiescence and neurogenesis. Here we show that human LINE1 RNA is essential for self-renewal and identity of human embryonic stem cells (hESCs). Silencing of LINE1 using either antisense oligonucleotides or CRISPR interference in naive hESCs leads to a strong induction of 8C-like cells (8CLCs). We found that genes derepressed upon LINE1 KD are not uniformly distributed across the genome, with an enrichment for chromosome 19, which includes key markers of the 8C state such as TPRX1. Silencing of TPRX1, but not other putative 8C regulators p53 or H3.XY, suppresses the induction of the 8C program in LINE1 KD hESCs. We found that LINE1 RNA is preferentially localized to the lamina and periphery of the nucleolus in hESCs. Sequencing of Lamina-Associated Domains (LADs) and Nucleolus-Associated Domains (NADs) reveals a preferential association of chromosome 19 with NADs in hESCs. However, 8CLCs have a distinct nucleolar morphology and a lower association of chromosome 19 and TPRX1 loci with the nucleolus relative to naive and primed hESCs, suggesting a role for nucleolar dynamics in the 8CLC-hESC transition. In agreement, LINE1 KD leads to disruption of nucleolar architecture with signs of nucleolar stress. Independent perturbations of the nucleolus induce the 8C program in hESCs. Genes induced by LINE1 KD are enriched for targets of Polycomb Repressive Complex (PRC2), and inhibition of PRC2 leads to a strong induction of 8C genes. Our results indicate that LINE1 coordinates nuclear compartmentalization and chromatin-mediated gene repression to prevent developmental reversion of hESCs. HighlightsO_LIKnockdown of LINE1 induces TPRX1-dependent emergence of 8C-like cells in hESCs. C_LIO_LIGenes de-repressed upon LINE1 KD are enriched for Chr 19 and PRC2 targets. C_LIO_LI8CLCs display dissociation of Chromosome 19 from the nucleolus. C_LIO_LIDisruption of the nucleolus or inhibition of PRC2 strongly induce the 8C program. C_LI

developmental biology↗

bioRxiv2024

Chromatin Remodelling in Damaged Intestinal Crypts Orchestrates Redundant TGFβ and Hippo Signalling to Drive Regeneration

Cell state dynamics underlying regeneration are under-characterized. Intestinal damage prompts reprogramming into revival stem cells (revSCs) that reconstitute Lgr5+ intestinal stem cells (ISCs). Single nuclei multiomics of chromatin accessibility and transcriptomes during regeneration from irradition showed revSCs display epigenetic profiles shared with ISCs and differentiated lineages. Furthermore, while revSC genes are accessible throughout homeostatic epithelia, damage-induced global alterations in crypt and revSC chromatin converge on TGF{beta}, as well as Hippo pathways. We show TGF{beta} directly induces functional revSCs and demonstrate individual revSCs form organoids with reconstituted Lgr5+ ISCs. Despite this, loss of TGF{beta} signalling yielded mild regenerative defects. In contrast, interference in both Hippo and TGF{beta} abolished revSCs, precluded generation of new ISCs and led to rapid intestinal collapse. Thus, the epithelium is poised to engage the revSC regenerative program that relies on crypt-localized, transient morphogen cues that function in a compensatory manner to support intestinal regeneration.

molecular biology↗

bioRxiv2021

Loss of epigenetic regulation disrupts lineage integrity, reactivatesmultipotency and promotes breast cancer

Systematically investigating the scores of genes mutated in cancer and discerning real drivers from inconsequential bystanders is a prerequisite for Precision Medicine, but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent long-tail breast cancer genes, which revealed epigenetic regulation as major tumor suppressive mechanism. We report that core or accessory components of the COMPASS histone methylase complex including KMT2C, KDM6A, BAP1 and ASXL2 ("EpiDrivers") cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that Cre-mediated activation of PIK3CAH1047R elicited an aberrant alveolar lactation program in luminal cells, which was exacerbated upon loss of EpiDrivers. Remarkably, EpiDriver loss in basal cells also triggered an alveolar-like lineage conversion and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. As EpiDrivers are mutated in 39% of human breast cancers, lineage infidelity and lactation mimicry may significantly contribute to early steps of breast cancer progression. Statement of significanceInfrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In-vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of lactation genes as potential early events in tumorigenesis.

cancer biology↗

bioRxiv2020

Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies - 2.1x105 copies of viral Orf1ab/l. However, the BGI detection system is [~]4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were [~]10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.

microbiology↗

bioRxiv2020

Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human specific cortical development and pathophysiology in an experimentally tractable system. While diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent (ie uniform) hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single cell profiling, that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

neuroscience↗