Ultrastructural localisation of protein interactions using conditionally stable nanobodies
| Autor: | Nick Martel, Emma Sierecki, Yann Gambin, Nicholas Ariotti, Nichole Giles, Thomas E. Hall, James Rae, Robert G. Parton |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Cell Membranes Gene Expression Plasma protein binding Biochemistry Green fluorescent protein Cell-free system Ascorbate Peroxidases Genes Reporter Gene expression Protein Interaction Mapping Electron Microscopy Biology (General) Staining Microscopy Protein Stability General Neuroscience Physics Methods and Resources Condensed Matter Physics Single Molecule Imaging Physical Sciences Cellular Structures and Organelles General Agricultural and Biological Sciences Protein Binding Proteasome Endopeptidase Complex QH301-705.5 Recombinant Fusion Proteins Green Fluorescent Proteins Biology Research and Analysis Methods General Biochemistry Genetics and Molecular Biology Protein–protein interaction Cell Line 03 medical and health sciences Cricetulus Electron Density Animals Protein Interactions Cytoplasmic Staining General Immunology and Microbiology Cell-Free System Biology and Life Sciences Proteins Membrane Proteins Protein Complexes Proteasomes Epithelial Cells Cell Biology Single-Domain Antibodies Apex (geometry) Luminescent Proteins Microscopy Electron 030104 developmental biology Membrane protein Proteasome Microscopy Fluorescence Coated Pits Specimen Preparation and Treatment Biophysics |
| Zdroj: | PLoS Biology, Vol 16, Iss 4, p e2005473 (2018) PLoS Biology |
| ISSN: | 1545-7885 1544-9173 |
| Popis: | We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. Author summary The use of enzymatic tags such as the ascorbate peroxidase (APEX) for electron microscopic detection of proteins is changing electron microscopy (EM) in the same way that the use of GFP and related proteins caused a revolution in light microscopy. We previously developed expression plasmids encoding GFP-binding peptide (or nanobody) fused to APEX, which allows EM localisation of GFP-tagged proteins in vivo. Here, we have generated conditionally stable GFP- and mCherry-binding nanobodies fused to APEX. Using co-transfection of these APEX nanobodies with fluorescent-tagged constructs, we recruit APEX and detect the tagged proteins by electron microscopy. As unbound conditionally stable nanobodies are efficiently degraded by the proteasome, the signal to noise ratio is dramatically reduced. This enables detection of less abundant proteins and eliminates the need to balance expression levels between fluorescent-labelled and APEX nanobody constructs. Furthermore, and perhaps most exciting, is our application of this method to bimolecular fluorescence complementation—in which two tagged proteins interact—allowing the detection and localisation of protein-protein interactions in EM. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
| Nepřihlášeným uživatelům se plný text nezobrazuje | K zobrazení výsledku je třeba se přihlásit. |