ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats DOI Creative Commons
Lukas Frank, Anne Rademacher, Norbert Mücke

et al.

Nucleic Acids Research, Journal Year: 2022, Volume and Issue: 50(11), P. e61 - e61

Published: Feb. 5, 2022

Alternative lengthening of telomeres (ALT) occurs in ∼10% cancer entities. However, little is known about the heterogeneity ALT activity since robust detection assays with high-throughput situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate single cells from accumulation single-stranded telomeric DNA and RNA. It involves one-step fluorescent hybridization approach followed by fluorescence microscopy imaging. Our reliably identified cell lines different tumor entities was validated three established models induction suppression. Furthermore, successfully applied ALT-FISH spatially resolve primary tissue sections leiomyosarcoma neuroblastoma tumors. Thus, our assay provides insights into tumors suited for applications, which will facilitate screening ALT-specific drugs.

Language: Английский

Spatial multi-omic map of human myocardial infarction DOI Open Access
Christoph Kuppe, Ricardo O. Ramirez Flores, Zhijian Li

et al.

Nature, Journal Year: 2022, Volume and Issue: 608(7924), P. 766 - 777

Published: Aug. 10, 2022

Language: Английский

Citations

388
FISH-quant v2: a scalable and modular tool for smFISH image analysis DOI Open Access

Arthur Imbert,

Wei Ouyang, Adham Safieddine

et al.

RNA, Journal Year: 2022, Volume and Issue: 28(6), P. 786 - 795

Published: March 28, 2022

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule fluorescence situ hybridization (smFISH) widely used single-cell-single-molecule imaging technique enabling quantitative studies its regulatory mechanisms. Today, these methods are applicable at large scale, which turn come with need for adequate tools data analysis exploration. Here, we present FISH-quant v2, highly modular tool accessible both experts non-experts. Our user-friendly package allows the user to segment nuclei cells, detect isolated RNAs, decompose dense clusters, quantify patterns visualize results single-cell level variations within cell population. This was validated applied on large-scale smFISH image sets, revealing diverse subcellular surprisingly high degree cell-to-cell heterogeneity.

Language: Английский

Citations

93
OME-Zarr: a cloud-optimized bioimaging file format with international community support DOI Creative Commons
Josh Moore, Daniela Basurto-Lozada, Sébastien Besson

et al.

Histochemistry and Cell Biology, Journal Year: 2023, Volume and Issue: 160(3), P. 223 - 251

Published: July 10, 2023

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals institutes across diverse modalities facing these have designed specification process (OME-NGFF) address needs. This paper brings together wide range those members describe cloud-optimized itself-OME-Zarr-along with tools data resources available today increase FAIR access remove barriers in scientific process. The current momentum offers an opportunity unify key component domain-the that underlies so many personal, institutional, global management analysis tasks.

Language: Английский

Citations

63
mRNA stability and m6A are major determinants of subcellular mRNA localization in neurons DOI Creative Commons
Inga Loedige, Artem Baranovskii, Samantha Mendonsa

et al.

Molecular Cell, Journal Year: 2023, Volume and Issue: 83(15), P. 2709 - 2725.e10

Published: July 13, 2023

For cells to perform their biological functions, they need adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model mRNA localization involves sequences called "zipcodes" that direct proper locations. However, while thousands localize cells, only a few zipcodes have been identified, suggesting additional mechanisms contribute localization. Here, we assess role stability by combining isolation soma neurites mouse primary cortical mESC-derived neurons, SLAM-seq, m6A-RIP-seq, perturbation destabilization mechanisms, analysis multiple datasets. We show depletion elements, such as m6A, AU-rich suboptimal codons, functions mechanism mediates associated with housekeeping several types neurons.

Language: Английский

Citations

45
Massively parallel identification of mRNA localization elements in primary cortical neurons DOI Creative Commons
Samantha Mendonsa, Nicolai von Kügelgen,

Sayaka Dantsuji

et al.

Nature Neuroscience, Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 16, 2023

Abstract Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of mRNAs specific areas, where they are translated into proteins with local functions. This mRNA is mediated by cis -regulatory elements mRNAs, commonly called ‘zipcodes’. Although there hundreds localized only a few zipcodes have been characterized. Here we describe novel neuronal zipcode identification protocol (N-zip) that can identify across 3′ untranslated regions. approach combines method separating principal neurons—cell bodies neurites—with massively parallel reporter assay. N-zip identifies let-7 binding site (AU) n motif as de novo mouse primary cortical neurons. Our analysis also provides, our knowledge, first demonstration an miRNA affecting suggests strategy for detecting more zipcodes.

Language: Английский

Citations

35
Age‐Associated Changes in Knee Osteoarthritis, Pain‐Related Behaviors, and Dorsal Root Ganglia Immunophenotyping of Male and Female Mice DOI

Terese Geraghty,

Alia M. Obeidat, Shingo Ishihara

et al.

Arthritis & Rheumatology, Journal Year: 2023, Volume and Issue: 75(10), P. 1770 - 1780

Published: April 25, 2023

Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age the strongest predictor development, and mechanisms driving are unclear. We undertook this study to characterize age-associated changes in knee OA, pain-related behaviors, dorsal root ganglion (DRG) molecular phenotypes mice both sexes.

Language: Английский

Citations

27
Deep-Tissue Spatial Omics: Imaging Whole-Embryo Transcriptomics and Subcellular Structures at High Spatial Resolution DOI Creative Commons
Valentina Gandin, Jun Kim,

Liang-Zhong Yang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 17, 2024

Summary The inherent limitations of fluorescence microscopy, notably the restricted number color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass conventional barrier. facilitates high-specificity, single-shot imaging per target for RNA protein species within thick specimens, mitigating molecular crowding issues encountered with other imaging-based omics techniques. We demonstrate whole-mount transcriptomics 254 genes an E6.5∼7.0 mouse embryo, achieving precise three-dimensional gene expression cell fate mapping across a specimen depth ∼ 310 µm. Utilizing expansion microscopy alongside cycleHCR, unveil complex network 10 subcellular structures primary embryonic fibroblasts. Furthermore, hippocampal slice, image 8 targets profile transcriptome 120 genes, uncovering gradients cell-type specific nuclear structural variances. provides unifying framework multiplex imaging, offering quantitative solution elucidating regulations deep tissue contexts research potentially diagnostic applications.

Language: Английский

Citations

11
A sense-antisense RNA interaction promotes breast cancer metastasis via regulation of NQO1 expression DOI Creative Commons
Bruce Culbertson, Kristle Garcia, Daniel Markett

et al.

Nature Cancer, Journal Year: 2023, Volume and Issue: 4(5), P. 682 - 698

Published: May 11, 2023

Abstract Antisense RNAs are ubiquitous in human cells, yet their role is largely unexplored. Here we profiled antisense the MDA-MB-231 breast cancer cell line and its highly lung metastatic derivative. We identified one RNA that drives progression by upregulating redox enzyme NADPH quinone dehydrogenase 1 (NQO1), named it NQO1-AS. Knockdown of either NQO1 or NQO1-AS reduced colonization a mouse model, investigation into indicated broadly protective against oxidative damage ferroptosis. Breast cells dependent on this pathway, dependence can be exploited therapeutically inducing ferroptosis while inhibiting NQO1. Together, our findings establish for regulating sense mRNA post-transcriptionally. Because predominantly affects females, disease models used study female origin results primarily applicable to females.

Language: Английский

Citations

20
Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID DOI Creative Commons
Andrea Bernardini, Pooja Mukherjee,

Elisabeth Scheer

et al.

Nature Structural & Molecular Biology, Journal Year: 2023, Volume and Issue: 30(8), P. 1141 - 1152

Published: June 29, 2023

Abstract Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure–function analyses, we show that human biogenesis occurs co-translationally. We discovered all protein heterodimerization steps happen during synthesis. identify TAF1—the largest complex—as a critical for assembly. TAF1 acts as flexible scaffold drives co-translational recruitment submodules preassembled cytoplasm. Altogether, our data suggest multistep hierarchical model culminates with assembly onto nascent polypeptide. envision this strategy could be shared other large complexes.

Language: Английский

Citations

18
M6A reduction relieves FUS-associated ALS granules DOI Creative Commons
Gaia Di Timoteo, Andrea Giuliani,

Adriano Setti

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: June 12, 2024

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated ALS pathogenesis, there formation of cytoplasmic inclusions produced by aggregation mutant proteins, among which RNA binding protein FUS. Here we show that, in neuronal cells and iPSC-derived MN expressing FUS, such are significantly reduced number dissolve faster when m

Language: Английский

Citations

7