Tracking surface glycans on live cancer cells with single-molecule sensitivity

Area of Science:

• Glycobiology
• Cell Biology
• Biophysics

Background:

• Cell surface glycans play crucial roles in cellular processes.
• Understanding glycan dynamics is essential for deciphering cell membrane functions.
• Previous methods limited the observation of individual glycans in live cells.

Purpose of the Study:

• To develop a method for dynamic single-molecule tracking and super-resolution imaging of cell surface glycans.
• To investigate the diffusion dynamics of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on live cell membranes.
• To visualize the structure of membrane nanotubes using super-resolution microscopy.

Main Methods:

• Metabolic labeling of glycans with isotopes.
• Bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) for probe conjugation.
• Controlled photobleaching of fluorescent probes for single-molecule localization.
• Stochastic optical reconstruction microscopy (STORM) for super-resolution imaging.
• Single-molecule tracking of labeled glycans in live mammary cancer cells.

Main Results:

• Achieved low spatial density of dye-labeled glycans for dynamic single-molecule tracking.
• Observed constrained diffusion for both N-linked sialic acids and O-linked GalNAc on live cell membranes.
• Interpreted constrained diffusion as intrinsic glycan mobility, not membrane immobilization.
• Utilized STORM to reveal the structure of dynamic membrane nanotubes.

Conclusions:

• The developed methodology enables unprecedented dynamic and super-resolution visualization of cell surface glycans.
• Constrained diffusion of glycans provides insights into their mobility and interactions within the plasma membrane.
• The study visualizes dynamic membrane nanotubes, offering new perspectives on cell structure and communication.