Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation
| Autor: | Emmi Pakarinen, Henri J. Huttunen, Maria Merezhko, Riikka-Liisa Uronen |
|---|---|
| Přispěvatelé: | Neuroscience Center, Institute of Biotechnology, Biosciences, Organotypic Vasculature Lab |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Time Factors GAMMA-SECRETASE Biosensing Techniques Proto-Oncogene Proteins c-fyn Biochemistry Organelles & Localization ALPHA-SECRETASE Amyloid beta-Protein Precursor Mice 0302 clinical medicine DOMAIN Protein-fragment complementation assay Luciferases PALMITOYLATION Lipid raft Research Articles biology Chemistry CHOLESTEROL Raft Gaussia princeps Protein subcellular localization prediction Cell biology AMYLOID PRECURSOR PROTEIN Protein Transport Recombinant Fusion Proteins Biophysics protein-fragment complementation assay high-throughput screening 03 medical and health sciences Membrane Microdomains Biochemical Techniques & Resources Palmitoylation Cell Line Tumor Animals Humans lipid microdomains Luciferase Molecular Biology MICRODOMAINS Lipid microdomain Cell Biology biology.organism_classification Peptide Fragments LIPID RAFTS RECEPTOR ACTIVATION 030104 developmental biology Microscopy Fluorescence 1182 Biochemistry cell and molecular biology Cell Membranes Excitation & Transport OVEREXPRESSION Proto-Oncogene Proteins c-akt 030217 neurology & neurosurgery |
| Zdroj: | Bioscience Reports |
| ISSN: | 1573-4935 0144-8463 |
| Popis: | The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called ‘lipid rafts’, in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts. |
| Databáze: | OpenAIRE |
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