Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Imaging

Background:

• Single molecule imaging in live cells is crucial for understanding protein interactions and dynamics in cellular signaling.
• Super-resolution localization imaging with stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) allows visualization of labeled molecules over time.
• Studying extended cellular responses requires high temporal and spatial resolution, often challenging with traditional imaging methods.

Purpose of the Study:

• To provide optimized experimental and analytical methods for quantifying protein interactions and dynamics in live cell membranes.
• To enable the study of protein interactions on biologically relevant distance and timescales using pair-correlation functions.
• To demonstrate the application of these methods in B cell receptor signaling.

Main Methods:

• Utilizing single molecule imaging in live cells with fluorophores exhibiting reversible dark states.
• Applying super-resolution localization microscopy for enhanced spatial resolution.
• Employing pair-correlation functions resolved in both space and time for quantitative analysis.
• Developing sample analysis code for post-processing of imaging data.

Main Results:

• Demonstrated a method to quantify protein interactions and dynamics in adhered live cell membranes.
• Successfully probed protein interactions on biologically relevant scales using spatiotemporal pair-correlation functions.
• Applied the approach to measure protein interactions within B cell receptor signaling pathways.

Conclusions:

• The presented methods are quantitative, sensitive, and broadly applicable to various cellular signaling systems.
• This approach overcomes limitations in temporal sampling for super-resolution imaging, enabling the study of dynamic processes.
• Advanced single molecule imaging provides unprecedented insights into molecular mechanisms underlying cell signaling.