Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins

Area of Science:

• Cell Biology
• Membrane Trafficking
• Epithelial Cell Biology

Background:

• Endocytosis is crucial for cellular uptake and is generally considered sensitive to N-ethylmaleimide (NEM).
• Polarized epithelial cells exhibit distinct apical and basolateral membrane domains with unique transport functions.

Purpose of the Study:

• To investigate the effect of NEM on endocytosis in polarized Madin-Darby canine kidney (MDCK I) cells.
• To elucidate the molecular mechanisms and membrane properties involved in NEM-modulated endocytosis in polarized cells.

Main Methods:

• Treatment of polarized MDCK I cells with NEM.
• Assessing endocytosis of ricin and horseradish peroxidase via apical and basolateral routes.
• Electron microscopy to visualize macropinosome formation.
• Investigating the involvement of phosphatidylinositol-3 kinase, protein kinase C, phospholipase D, and ATP.
• Analyzing ricin and transferrin endocytosis and recycling in the presence and absence of NEM.

Main Results:

• NEM treatment significantly increased apical endocytosis of ricin and horseradish peroxidase, inducing large apical macropinosomes.
• Apical endocytosis stimulation by NEM was dependent on phosphatidylinositol-3 kinase, protein kinase C, phospholipase D, and ATP.
• Unlike non-polarized cells, NEM allowed basolateral ricin endocytosis while blocking transferrin endocytosis.
• NEM did not inhibit the recycling of previously endocytosed ricin, indicating a NEM-resistant fusion machinery.

Conclusions:

• NEM differentially affects apical and basolateral endocytosis in polarized MDCK I cells, enhancing apical uptake.
• The findings suggest distinct signaling pathways and ATP-dependent mechanisms regulate NEM-stimulated apical endocytosis.
• Polarized MDCK I cell plasma membranes possess unique fusogenic properties compared to non-polarized cells, with a NEM-resistant fusion machinery for recycling.