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Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures.

Katharina B Beer | Gholamreza Fazeli | Kristyna Judasova | Linda Irmisch | Jona Causemann | Jörg Mansfeld | Ann M Wehman
Nature communications | 2019

Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we use a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes. These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.

Pubmed ID: 31375709

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Addgene (tool)

RRID:SCR_002037

Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.

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RRID:SCR_005375

THIS RESOURCE IS NO LONGER IN SERVICE. Documented on May 5,2022.Tool that predicts interactions between transcription factors and their regulated genes from binding motifs. Understanding vertebrate development requires unraveling the cis-regulatory architecture of gene regulation. PRISM provides accurate genome-wide computational predictions of transcription factor binding sites for the human and mouse genomes, and integrates the predictions with GREAT to provide functional biological context. Together, accurate computational binding site prediction and GREAT produce for each transcription factor: 1. putative binding sites, 2. putative target genes, 3. putative biological roles of the transcription factor, and 4. putative cis-regulatory elements through which the factor regulates each target in each functional role.

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RRID:SCR_008452

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RRID:SCR_008988

American chemical, life science and biotechnology company owned by Merck KGaA. Merger of Sigma Chemical Company and Aldrich Chemical Company. Provides organic and inorganic chemicals, building blocks, reagents, advanced materials and stable isotopes for chemical synthesis, medicinal chemistry and materials science, antibiotics, buffers, carbohydrates, enzymes, forensic tools, hematology and histology, nucleotides, proteins, peptides, amino acids and their derivatives.

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RRID:CVCL_0030

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RRID:SCR_002677

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RRID:SCR_007370

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RRID:SCR_003070

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RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

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RRID:SCR_014212

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RRID:SCR_002285

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RRID:SCR_013673

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RRID:SCR_014199

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HeLa Kyoto (cell line)

RRID:CVCL_1922

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