Small nanoparticles: Design and synthesis of shape variants as multiplex biological probes for immuno-electron microscopy.

Summary Electron microscopy (EM) is a core technique within cell biology research allowing nanometer (nm) scale analysis of cell structure. In order to visualise the precise location of specific proteins and lipids, electron dense labels conjugated to antibodies are used. Gold nanoparticles are preferred because of their easily tunable size in the range of 2-15 nm and simple conjugation to targetting antibodies. Typically, labelling within a sample (immuno-EM) is restricted to three targets using visually separable gold sizes (5-, 10- and 15 nm). Increasingly, cell biology research requires simultaneous analysis of many more components within for example signalling networks. In addition, certain analysis methods are limited to using gold within a very narrow size range, further reducing labelling options. We aim to adapt methods for synthesising gold shape variants (bars, cubes, triangular plates, octahedrons) to produce particles with a size range suitable for immuno-EM. At these small sizes (2-15 nm) spherical particles are the default, by increasing the suite of visually identifiable shapes we will significantly increase the labelling options for immuno-EM. Using technology developed in our laboratory, the particles can be irreversibly linked to almost any compound. We will bind the nanoparticles to antibodies targetted against components of an important pathway involved in regulating cell growth and survival - the Ras cascade. These components are localised on the cell surface and can be visualised using EM protocols developed in our laboratory for studying protein and lipid distributions within isolated cell surface fragments. Unfortunately, this approach is limited to using very small gold (5 nm or less), therefore the development of shape variants dramatically increases the number of components that can be simultaneously identified within a single sample. Our study aims use these tools to determine the extent to which components of the signalling pathway are compartmentalised i.e. found in discrete patches that help to improve signalling efficiency or specificity. Ras is a model protein for studying isoform specificity within cell signalling pathways and combined with our EM approach has provided insights into compartmentalisation of the cell surface, therefore the research has implications beyond the immediate field of Ras biology. In a broader sense, the new shape variants will benefit the entire cell biology community who currently rely on immuno-EM for their studies.

Objectives: to synthesize 2-20nm gold shape variants to functionalise and demonstrate utility for immuno-EM to investigate cell surface compartmentalisation Gold seed synthesis: Parameters such as reductant and ligand type and concentrations and reaction temperature will have to be optimised to produce sub-nanometer clusters. Growth reactions forming non-spherical shapes: Non-spherical particles evolve by the symmetry breaking of face-centered cubic metallic structures by preferential adsorption of capping ligands on different crystal faces to produce anisotropic shapes. By controlling the kinetics of growth of various facets of a seed, different shapes can be achieved. We will systematically study the effects of pH, temperature, initial concentration of Au seed and concentration ratio of Au seed to Au3+ in the growth solution, the nature and concentration of reducing agents (eg. ascorbate, sodium citrate), capping ligands (eg. CTAB, PVP), and silver nitrate as additives in controlling the size, shape, monodispersity and yield of nanoparticles. Protecting the nanocrystal and immuno-functionalisation: We will use an approach previously developed in our laboratory to protect nanocrystals from the Ostwald ripening process and functionalise them by incubating with the pentapeptide CALNN. Antibodies will be covalently conjugated to CALNN coated to the gold nanoparticles. Characterisation: To be conducted throughout using UV-vis-NIR spectrophotometry, TEM, Mass Spectroscopy, NMR and X-ray diffraction. Immuno-EM: Gold shape variants will be directly conjugated to antibodies specific to signalling proteins (eg. Ras) or variously tagged PH-domains specific for phosphoinositide species. Ultra-thin cryosections and isolated plasma membrane sheets will be prepared and immuno-labelled using standard protocols. We will examine cell surface Ras isoform and phosphoinositide distributions in stimulated and control HeLa cells.