研究目的
To visualize EphA4 activity in different compartments of the plasma membrane and investigate its regulation in time and space.
研究成果
The developed FRET-based EphA4 biosensor enables high spatiotemporal monitoring of EphA4 activity in live cells, revealing differential activation in membrane microdomains with stronger responses in non-raft regions, regulated by the actin cytoskeleton. This tool provides insights into EphA4 signaling dynamics and can be used for further investigations in development and disease.
研究不足
The study relies on biosensor design which may not fully capture all aspects of endogenous EphA4 signaling; detergent extraction methods for membrane studies are disruptive; potential for protein contamination in fractions; cell morphology changes could affect FRET measurements.
1：Experimental Design and Method Selection:
Developed a FRET-based biosensor using ECFP and YPet to detect EphA4 phosphorylation at Tyr596 and intramolecular binding to Src SH2 domain. Designed biosensors targeted to raft and non-raft membrane microdomains using lipid modifications (Lyn and KRas motifs).
2：Sample Selection and Data Sources:
Used purified biosensor proteins from E. coli for in vitro tests. Employed mammalian cell lines including HeLa, MEF, and HEK293AD cells expressing endogenous or exogenous EphA
3：List of Experimental Equipment and Materials:
Fluorescence plate reader (TECAN, Sapphire II), Nikon microscope with CCD camera, MetaFluor and MetaMorph software, various reagents such as EphA4 kinase, ephrin ligands, inhibitors (e.g., PP1, cytochalasin D), and cell culture materials.
4：Experimental Procedures and Operational Workflow:
Expressed and purified biosensors; performed in vitro kinase assays; transfected cells with biosensor plasmids; stimulated cells with pervanadate or ephrin ligands; imaged FRET responses using fluorescence microscopy; analyzed data with software.
5：Data Analysis Methods:
Measured ECFP/FRET emission ratios; normalized data to basal levels; used statistical methods (mean ± SEM) with multiple replicates.
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fluorescence plate reader
Sapphire II
TECAN
Measuring fluorescence emission ratios for FRET analysis in vitro
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microscope
Nikon
Imaging live cells for FRET measurements
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software
MetaFluor 6.2
Universal Imaging
Controlling image acquisition and analyzing FRET data
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software
MetaMorph
Universal Imaging
Image processing and analysis
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filter
420DF20
Excitation filter for fluorescence microscopy
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dichroic mirror
455DCXRU
Splitting light for FRET imaging
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emission filter
480DF40
Filter for CFP emission
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emission filter
535DF25
Filter for YFP emission
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Quickchange site-directed mutagenesis kit
Stratagene
Generating mutations in biosensor constructs
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pRSETB plasmid
Invitrogen
Cloning vector for bacterial expression
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pcDNA3 plasmid
Invitrogen
Cloning vector for mammalian cell expression
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Lipofectamine 2000
Invitrogen
Transfecting DNA plasmids into cells
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EphA4 kinase
Sigma
Phosphorylating the biosensor substrate in vitro
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c-Src kinase
Upstate
Testing biosensor specificity in vitro
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PDGFR
Sigma
Testing biosensor specificity in vitro
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lambda protein phosphatase
New England Biolabs
Dephosphorylating the biosensor to reverse FRET response
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trypsin
Gibco
Cleaving the biosensor to confirm FRET mechanism
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ephrinA3 Fc chimera
R&D systems
Stimulating EphA4 activation in cells
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ephrinA1 Fc chimera
R&D systems
Stimulating EphA4 activation in cells
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ephrinB2 Fc chimera
R&D systems
Stimulating EphA4 activation in cells
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