title: Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75(NTR).
abstract: Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75(NTR). However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75(NTR) in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75(NTR) can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.
authors.affiliation: The Lundbeck Foundation Research Center MIND, Aarhus University8000 Aarhus C, Denmark; Stereology and Electron Microscopy Laboratory & Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University8000 Aarhus C, Denmark; Department of Biomedicine, Aarhus University8000 Aarhus C, Denmark; Department of Clinical Medicine, Aarhus University8000 Aarhus C, Denmark.
authors.forename: Sune
authors.initials: S
authors.lastname: Skeldal
authors.affiliation: The Lundbeck Foundation Research Center MIND, Aarhus University8000 Aarhus C, Denmark; Stereology and Electron Microscopy Laboratory & Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University8000 Aarhus C, Denmark; Department of Clinical Medicine, Aarhus University8000 Aarhus C, Denmark.
authors.forename: Maj M
authors.initials: MM
authors.lastname: Kjaergaard
authors.affiliation: Fetal Programming of Disease Research Chair, Department of Zoology, College of Science, King Saud University Riyadh, Saudi Arabia.
authors.forename: Saleh
authors.initials: S
authors.lastname: Alwasel
authors.affiliation: The Lundbeck Foundation Research Center MIND, Aarhus University8000 Aarhus C, Denmark; Stereology and Electron Microscopy Laboratory & Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University8000 Aarhus C, Denmark; Department of Clinical Medicine, Aarhus University8000 Aarhus C, Denmark.
authors.forename: Jens R
authors.initials: JR
authors.lastname: Nyengaard
keywords: FRET-based fluorescence plate reader assay; high throughput; p75NTR; proNGF; sortilin
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