Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways

Area of Science:

• Biochemistry
• Cell Biology
• Drug Discovery

Background:

• Cyclotides are plant-derived cyclic peptides with inherent stability and biological activities.
• Their unique structure and stability make them promising candidates for drug development.
• Understanding their cellular internalization is crucial for therapeutic applications.

Purpose of the Study:

• To investigate the cellular uptake mechanisms of the cyclotide MCoTI-I in live HeLa cells.
• To determine the pathways involved in MCoTI-I internalization.
• To assess the potential for MCoTI-I re-targeting.

Main Methods:

• Real-time confocal fluorescence microscopy imaging of live HeLa cells.
• Temperature-dependent uptake studies.
• Colocalization studies with endocytosis markers (10K-dextran, cholera toxin B, EGF).
• Inhibition studies using Latrunculin B and EIPA.

Main Results:

• MCoTI-I is readily internalized by HeLa cells in a temperature-dependent manner.
• Primary uptake occurs via fluid-phase endocytosis, with contributions from cholesterol-dependent and clathrin-mediated pathways.
• Macropinocytosis is not the sole pathway; combined inhibition reduced uptake by approximately 80%.
• MCoTI-I accumulates in late endosomal and lysosomal compartments with directed vesicle movement.

Conclusions:

• MCoTI-I utilizes multiple, dominant endocytic pathways in HeLa cells.
• The cyclotide gains access to general endosomal/lysosomal pathways.
• MCoTI-I can be potentially re-targeted to specific receptors by adding targeting ligands.