研究目的
To develop an optogenetically regulated membrane receptor-ligand pair for precise control of integrin-matrix interactions using light.
研究成果
The OptoIntegrin-OptoMatrix system successfully enables light-controlled cell-matrix interactions and activation of downstream mechanosensory pathways, providing a novel tool for studying integrin signaling with high spatiotemporal precision.
研究不足
The system is specific to the engineered integrin and matrix pair; it may not fully replicate natural integrin signaling complexities. Optimization is needed for broader applicability to other receptor-ligand pairs.
1：Experimental Design and Method Selection:
The study designed an optogenetic system using phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) from Arabidopsis thaliana to control integrin-matrix interactions. The rationale was to enable light-inducible and reversible binding.
2：Sample Selection and Data Sources:
Human cell lines (HEK-293T, HeLa, MCF7) were used, stably expressing engineered integrins. Data were acquired through flow cytometry, microscopy, and western blotting.
3：List of Experimental Equipment and Materials:
Includes glass slides, LEDs for illumination, antibodies for staining, and recombinant proteins like PhyB1-
4：Experimental Procedures and Operational Workflow:
6 Cells were transfected or transduced with integrin constructs, seeded on PhyB-coated matrices, illuminated with 660 nm or 740 nm light, and analyzed for attachment, spreading, and signaling pathway activation.
5：Data Analysis Methods:
Statistical analyses used Cochran-Mantel-Haenszel test, Chi-square test, and Welch-test; imaging data were processed with Fiji and OpenCV software.
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LED
LED660N-03
Roithner LaserTechnik
Illumination at 660 nm wavelength for activating the optogenetic system.
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LED
H2A1-H740
Roithner LaserTechnik
Illumination at 740 nm wavelength for deactivating the optogenetic system.
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LED
LED740-01AU
Roithner LaserTechnik
Illumination at 740 nm wavelength.
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Microscope
Axio Observer Z1/7
Zeiss
Imaging of cells during experiments.
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Spectrometer
AvaSpec-ULS2048
Avantes BV
Measurement of light intensities and spectra.
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Confocal Microscope
Eclipse Ni-E with C2 confocal laser scanner
Nikon Instruments
Confocal imaging for immunostaining analysis.
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Antibody
A2102
Thermo Fisher
Secondary antibody Alexa Fluor 488-labelled donkey anti-mouse IgG for fluorescence detection.
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Antibody
A-21244
Thermo Scientific
Secondary antibody Alexa Fluor 647-labelled goat anti-rabbit IgG for fluorescence detection.
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NeutrAvidin
31050
Thermo Scientific
Coating agent for glass slides to bind biotinylated PhyB1-651.
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LED
LH W5Am
Osram Opto Semiconductors
Illumination at 660 nm wavelength.
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LED
L-53SR
Kingbright Electronic
Illumination at 660 nm wavelength.
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LED
LZ4-00R308
LED Engin
Illumination at 740 nm wavelength.
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Microscope
EVOS Xl
Fisher Scientific
Transmitted light microscopy for cell imaging.
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Flow Cytometer
GALLIOS
Beckman Coulter
Analysis of cell fluorescence and surface expression.
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Cell Sorter
S3e
Bio-Rad
Fluorescence-activated cell sorting for selecting high-expressing cells.
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Imaging System
ImageQuant LAS-4000 mini
GE Healthcare
Detection of chemiluminescence in western blotting.
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Glass Bottom Dish
81158
IBIDI
Container for live cell imaging experiments.
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Dialysis Tube
SnakeSkin
Fisher Scientific
Dialysis of proteins like PhyB1-651.
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Antibody
H9658
Sigma
Primary antibody for detecting HA tag in western blotting and flow cytometry.
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Antibody
HPA051309
Atlas Antibodies
Primary antibody for paxillin staining.
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