Development of gradient guidance assays using nanogold surface patterning

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DESCRIPTION (provided by applicant): Most sensory input to the brain is mapped topographically, with nearest neighbor relationships of the projecting neurons maintained in their connections within target areas. For example, retinal ganglion cell (RGC) axons project topographically to retinal targets in the brain, allowing visual images to be transferred in a spatially intact form. It is believed that topographic maps form using gradients of axon guidance molecules expressed in both projection and target neurons. Although there is a consensus that gradients are used in topographic mapping, many questions and hypotheses about how axons read molecular gradients and branch at their appropriate sites remain unknown. This proposal describes a new method for fabricating density gradients of biomolecules on solid substrates. The approach employs a hierarchical strategy in which proteins are first attached to nanometer-sized metal particles, and the resulting protein-nanoparticle bioconjugates are subsequently assembled from solution onto a substrate surface. The resulting surface topography can be characterized by monitoring the large and distinctive visible light absorptions of metal nanoparticles by imaging the nanoparticles with atomic force microscopy. This method of nanoscale surface engineering is capable of forming molecular gradients of varying concentration and slope, in one and two- dimensions, and does not require expensive or sophisticated equipment. Moreover, because gold and silver nanoparticles absorb visible light of different energies, surface density gradients containing two different proteins on a single substrate may be assembled and characterized. Two dimensional protein patterning will increase the complexity of in vitro cell-based assays significantly. The utility of surfaces containing protein density gradients will be illustrated through studies of topographic mapping in the visual system. One and two-dimensional surface gradients of EphA receptors and ephrin-A ligands will be constructed in a manner that recapitulates their graded expression during visual system development. Surface-bound neuronal growth assays will deepen our understanding of the molecular mechanisms of axon guidance by more closely mimicking protein patterning encountered in living systems. This understanding will not only allow us to understand how neural connections are made in the brain but will also guide us to be able to rewire connections after injury.