Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling

Area of Science:

• Molecular biology
• Cell biology
• Biophysics

Background:

• Imaging and tracking single molecules in live cells is challenging due to limitations in resolution and molecule crowding.
• Precisely controlling the number of labeled molecules is crucial for accurate observation of cellular processes.

Purpose of the Study:

• To develop a universal genetic strategy for precise control of fluorescently labeled molecule copy numbers in cells.
• To enhance the capabilities of live-cell single-molecule tracking for extended durations and broader applications.

Main Methods:

• A novel universal genetic system was engineered to regulate the cellular copy number of fluorescently labeled molecules.
• The system achieves a dynamic range of approximately 10,000-fold, allowing for sparse labeling of proteins with varying abundance.
• Combined with photostable labels, this method significantly extends the time frame for automated single-molecule tracking.

Main Results:

• Demonstrated long-term imaging of synaptic vesicle dynamics in cultured neurons and intact zebrafish.
• Identified a "waterfall" mechanism in the axon initial segment governing synaptic vesicle transport polarity.
• Observed transcription factors exhibiting hop-diffusion behavior between clustered binding sites within specific nuclear regions.

Conclusions:

• The developed genetic strategy dramatically expands the spatiotemporal scales for live-cell single-molecule measurements.
• This advancement facilitates quantitative investigations into the complex control of molecular dynamics in vivo.
• Revealed novel insights into synaptic vesicle transport and gene regulation mechanisms within the nucleus.