Dj Brayshaw

Optimisation of sample preparation methods for air imaging of ocular mucins by AFM.

The addition of cations to the imaging buffer for AFM has been previously shown to improve the binding of biological molecules to mica. Investigations were carried out to find the concentration of NiCl(2) required to immobilize mucin molecules on a freshly cleaved mica surface, for imaging using intermittent contact in air. Drop-deposition of samples prepared in HEPES buffer with 1, 2 and 5mM NiCl(2) revealed the sensitivity of the mucin molecules to salt. Dialysis of the mucin solutions dramatically reduced the amount of salt present and allowed single molecules to be imaged, revealing a variation in thickness along their length. Spray deposition of the same mucin solutions produced single molecules that, although less affected by co-adsorbed salt, showed a degree of self-folding. This shows the sensitive balance between HEPES and NiCl(2) required for successful imaging of the sub-molecular features of individual mucin molecules.

Dynamic molecular resolution imaging of preocular fluid impressions

Aim: The preocular fluid is renewed with molecules secreted by the underlying cells and with lacrimal gland secretions, while maintaining a stable surface topography. The authors tested the hypothesis that interactions between gelled and newly inserted mucins are the key to this stability. Methods: Using atomic force microscopy, the authors studied the topography of the freshly isolated preocular fluid obtained by impression cytology. The effects of adding mucins to this impression were compared with adding mucins to a pure mucin macromolecular assembly as a single component control to the more complex preocular fluid. The control structure was built up by repeated addition of pure ocular mucin to a tethering surface. Results: Imaging at molecular resolution showed a thin layer of superficial preocular fluid with an appearance consistent with a gel that was very flat, with surface roughness of approximately 0.1 nm. Mucin molecules adhering to a clean flat surface maintained their individual character when overlapping, whereas molecules integrating in the impression could not be followed individually. Both the preocular impression and the pure mucin assembly were stable under imaging for at least 90 minutes. The roughness of the pure mucin network decreased as more mucin was added. In contrast, there was a small increase in the roughness of the 2.25 μm2 area of impression over the 60 minutes of continuous imaging, although locally there appeared to be infill of low height features. Disulphide bond breaking resulted in the collapse of the imaged structure in both the pure mucin control and the more complex ex vivo preocular impression. Conclusions: Polymeric mucins linked by disulphide bonds prevent or lessen loss of ocular surface material into the surrounding aqueous tears.

Reducing a polymer to its subunits as an aid to molecular mapping

Detailed, single-molecule AFM mapping can further structural studies of polymeric biomolecules by pinpointing discrete changes in subunits or subunit concatenation. This study explored the binding of purified (ocular) mucins, polymers composed of genetically identical subunits, to controlled surfaces. This process was followed in situ, in real time, as were the effects of the disulfide bond reducing agent dithiothreitol (DTT), a reagent routinely used to depolymerize mucins. The addition of this reagent, while mucins were bound to gold surfaces by thiol-type binding, suggested a way of assessing the strength and extent of this gold–molecule bond formation relative to other forms of mucin–substrate interactions. Real-time AFM has allowed us to visualize the cleavage of in-chain disulfide bonds in a single mucin molecule, and subsequent removal from the substrate of mucin subunits between disulfide sites. In contrast, mucins that were covalently bound via amine groups to a self-assembled succinimide monolayer were not observed to move from the point of their initial attachment to the substrate and the addition of dithiothreitol was not followed by the loss of any sections of molecules from the substrate, emphasizing the different immobilization bond types. This demonstration of the ability to follow the structural changes to a single molecule as a result of a series of chemical processes points to new approaches to single-molecule mapping, and localization of specific chemical moieties.