研究目的
Investigating the chromatin-binding kinetics of the transcription factor SRF at single-molecule resolution upon cell stimulation.
研究成果
SRF exhibits three distinct chromatin residence time regimes (short, intermediate, long) that are modulated by cell stimulation, with increased long binding times and fractions upon stimulation, mediated by actin signaling, MAP kinases, and MRTF cofactors. This reveals highly dynamic TF-chromatin interactions.
研究不足
The study is limited to fibroblasts and neurons; other cell types were not examined. The temporal resolution may not capture very rapid changes, and the use of fusion proteins could potentially affect native SRF function, though controls showed minimal interference.
1：Experimental Design and Method Selection:
Used single-molecule tracking (SMT) with HaloTag fusion proteins and light-sheet microscopy (HILO) to observe SRF dynamics in live cells.
2：Sample Selection and Data Sources:
Employed murine NIH 3T3 fibroblasts and primary hippocampal neurons from mice, with stable expression of Halo-SRF.
3：List of Experimental Equipment and Materials:
Included custom-built fluorescence microscope with Nikon TiE body, Toptica 638-nm laser, Cobolt 515-nm laser, Nikon 100×
4：45 Plan Apo objective, Andor iXon Ultra DU 897U EMCCD camera, Bioptechs heatable culture dishes, Promega TMR and SiR ligands, and various inhibitors like Latrunculin B and UExperimental Procedures and Operational Workflow:
Cells were labeled with fluorophores, starved, stimulated with serum or BDNF, and imaged with varying illumination schemes (e.g., time-lapse and interlaced time-lapse microscopy) to track molecule binding and diffusion.
5：Data Analysis Methods:
Used tracking software for single-molecule analysis, fitted data with multi-component diffusion and decay models, and performed statistical tests like Mann-Whitney u test and t-test.
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laser
IBEAM-SMART-640-S
Toptica
638-nm laser for exciting SiR-labeled cells.
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camera
iXon Ultra DU 897U
Andor
EMCCD camera for detecting fluorescence.
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microscope
TiE
Nikon
Used for single-molecule imaging with HILO microscopy.
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laser
Cobolt Jive
Cobolt
515-nm laser for exciting TMR-labeled cells.
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objective
100× 1.45 Plan Apo
Nikon
High-NA objective for HILO microscopy.
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culture dish
ΔT culture dishes
Bioptechs
Heatable culture dishes with glass bottoms for live-cell imaging.
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controller
Delta T5 μ-Environmental Culture Dish Controller
Bioptechs
Controls temperature during imaging.
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software
NIS Elements
Nikon
Controls the microscope setup.
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data acquisition card
NIDAQ
National Instruments
Used for data acquisition in the setup.
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TMR ligand
Promega
Fluorophore for labeling HaloTag fusion proteins.
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SiR ligand
Promega
Fluorophore for labeling HaloTag fusion proteins.
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RNA isolation kit
ISOLATE II RNA/DNA/Protein kit
Bioline
Used for isolating total RNA.
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reverse transcriptase
Promega
Used for reverse transcription in qPCR.
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qPCR machine
Light Cycler 480II
Roche
Used for quantitative real-time PCR.
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qPCR master mix
Power SYBR green PCR master mix
Takara
Used in qPCR reactions.
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ChIP antibody
anti-HALO
Promega
Rabbit polyclonal antibody for ChIP assays.
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ChIP antibody
IgG antibody
Santa Cruz
Rabbit polyclonal control antibody for ChIP.
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PCR purification kit
Qiagen
Used for purifying DNA after ChIP.
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