Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells

Area of Science:

• Cell Biology
• Molecular Biology
• Endocytosis Research

Background:

• Macropinocytosis is a key cellular process for fluid-phase uptake, characterized by membrane ruffling.
• High-molecular weight dextran is a standard marker for macropinocytosis due to its inability to enter smaller endocytic vesicles.
• CYFIP-related Rac1 Interactor (CYRI) proteins have recently been implicated in regulating macropinocytosis.

Purpose of the Study:

• To detail a protocol for detecting internalised dextran in cultured cells.
• To present an image analysis pipeline for quantifying dextran uptake using ImageJ/Fiji.
• To investigate the role of CYRI proteins in macropinocytosis.

Main Methods:

• Utilizing an image-based dextran uptake assay with high-molecular weight dextran (≥70kDa).
• Culturing cells and applying experimental treatments to assess macropinocytosis.
• Employing ImageJ/Fiji software for image acquisition and analysis of internalised dextran.

Main Results:

• Cells lacking CYRI proteins internalised significantly less dextran compared to wild-type cells.
• The developed protocol allows for quantitative assessment of dextran uptake.
• The image analysis pipeline enables efficient and automatable processing of experimental data.

Conclusions:

• CYRI proteins play a significant role in facilitating macropinocytosis.
• The described protocol and analysis pipeline are valuable tools for studying macropinocytosis.
• This methodology is adaptable for various experimental conditions and treatments affecting endocytosis.