Tracking single molecules in the live cell plasma membrane-Do's and Don't's

Area of Science:

• Cell Biology
• Biophysics
• Optical Microscopy

Background:

• High-sensitivity microscopy now allows resolving single, fluorescence-labeled biomolecules within living cells.
• Techniques developed in the late eighties for tracking labeled biomolecules paved the way for current advancements.
• Millisecond time-resolution imaging of weakly fluorescent structures is now state-of-the-art.

Purpose of the Study:

• To describe strategies for utilizing single molecule trajectories to deduce nanoscopic structures in live cells.
• To focus on elucidating plasma membrane organization using single molecule tracking.
• To provide a framework for interpreting molecular diffusion patterns within the plasma membrane.

Main Methods:

• Single molecule tracking of fluorescently labeled membrane constituents (proteins, lipids).
• Analysis of molecular trajectories to identify diffusion behaviors (free vs. subdiffusive).
• Application of analytical models to interpret diffusion data and infer membrane organization.

Main Results:

• Single molecule trajectories reveal complex interactions influencing molecular diffusion.
• Subdiffusive behavior indicates stronger or correlated interactions experienced by membrane components.
• This approach provides insights into nanoscopic membrane structures like rafts, microdomains, fences, and pickets.

Conclusions:

• Single molecule tracking offers a powerful method to study live cell plasma membrane organization at the nanoscopic level.
• Interpreting diffusion patterns provides crucial information about molecular interactions and membrane architecture.
• Awareness of potential analytical pitfalls is essential for accurate quantitative and qualitative interpretations.