Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy

Background:

• Macropinocytosis is a crucial cellular process for nutrient uptake, yet its mechanisms remain incompletely understood.
• Conventional imaging methods struggle to capture dynamic events like macropinosome formation and maturation.
• Fluorescent dextrans are common markers for studying endocytic pathways.

Purpose of the Study:

• To develop and optimize live-imaging techniques for visualizing macropinocytosis.
• To improve the segmentation and quantification of macropinosomes.
• To demonstrate advanced imaging strategies for studying cellular uptake.

Main Methods:

• Live imaging of cells in high concentrations of fluorescent dextran to visualize unlabeled cells in relief.
• Dual-labeling with different sized dextrans and co-expression of lipid probes/fluorescent proteins.
• Correlative light and electron microscopy (CLEM) for ultrastructural detection of internalized dextran.

Main Results:

• The optimized method effectively visualizes cell surface ruffles, macropinosome formation, and internalized macropinosomes.
• Dual-labeling and co-expression allow for clear demarcation of macropinosomes and other endosomes.
• CLEM successfully detected internalized dextran at the ultrastructural level.

Conclusions:

• The described live-imaging approaches significantly enhance the visualization and analysis of macropinocytosis.
• These methods are adaptable to various experimental systems and imaging modalities, including 3D.
• The optimized techniques facilitate a deeper understanding of cellular fluid-phase uptake.