Atomic Force Microscopy of Ocular Mucins Sequential mapping and manipulation of a single molecule
The aim of the project is to isolate and purify mucins from ocular cell lines, and map
individual molecules using the Atomic Force Microscope (AFM) to ultimately create a
recognition image as well as a topographical one.
The ocular surface is covered in a complex viscous secretion called mucus which prevents
desiccation of the underlying epithelium, acts as a lubricant during blinking, and provides
a barrier against pathogens. Mucus is composed primarily of water, but the main component
responsible for the visco-elastic gel-like properties is the glycoconjugate mucin. Mucins are
comprised of a protein backbone with a central tandem repeat region of aminoacids rich in serine,
threonine and proline residues decorated by O-linked sugar side chains.
Using flexible linkers, AFM tips were functionalized with various antibodies and lectin molecules
which bind specifically to epitopes on the mucin molecules. These will recognize either the N or
C-terminus of the peptide core, or will bind to the sugar side chains. Control tips have been
functionalized using antibody-blocking peptide conjugates. The AFM can be used in force-volume
mode to produce simultaneous topographic and force extension curves. This will highlight the
localisation, number and specific energies of recognition bonds.
Figure 1. AFM image of mucins in air. |
Figure 2. AFM image of mucins in liquid. |