Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Endocytosis is a vital cellular process in all eukaryotic cells.
• Live cell imaging has been instrumental in understanding endocytosis mechanisms.
• Existing imaging assays may require prior hypotheses about vesicle dynamics.

Purpose of the Study:

• To detail the pulsed-pH (ppH) protocol for high-resolution live cell imaging of endocytosis.
• To explain how the ppH protocol quantifies endocytic activity and protein recruitment in real time.
• To provide a comprehensive procedure for implementing the ppH protocol.

Main Methods:

• Utilizes a pH-sensitive fluorescent protein (e.g., superecliptic pHluorin) linked to a cargo protein.
• Employs fast perfusion systems to rapidly switch extracellular solutions between pH 7.4 and 5.5 every 2 seconds.
• Involves transient transfection for reporter protein expression, synchronized live cell image acquisition, and automated data analysis.

Main Results:

• Achieves an unparalleled temporal resolution of 2 seconds for detecting individual endocytic vesicles (EVs).
• Quantifies endocytic activity and associated protein dynamics in living cells without requiring a priori assumptions.
• Provides a 2D quantitative analysis of the endocytic process.

Conclusions:

• The ppH protocol offers a robust and versatile method for studying endocytosis with high temporal resolution.
• This technique allows for unbiased investigation of vesicle formation and protein dynamics during endocytosis.
• The detailed procedure facilitates the application of ppH for diverse endocytosis research.