The long term objective of this proposed research is to understand the developmental role of carbohydrate recognition during the multiple steps of synapse formation. Because neurons lose their specific carbohydrate markers upon deafferentation, we have developed the leech as a model system in which we can experimentally test the developmental function of carbohydrate recognition in an intact nervous system. Previously, we have demonstrated that different carbohydrate markers mediate sequential steps in axonal targeting. A mannose-specific recognition mediates the initial defasciculations and dispersal of axons across the target. In contrast, recognition via carbohydrate markers on complex/hybrid type glycoconjugates leads to the subsequent assembly of different axonal subset to their restricted target regions. In this proposed work we will characterize these developmentally important carbohydrate markers structurally by correlating chemical information, gained from combining methods of mass spectrometry and NMR spectroscopy, immunopurification and exoglycosidase digestion, with data on the markers' terminal glycosyl residues gained in functional assays. We will also expand our knowledge on the receptors to which these carbohydrate markers bind during axonal targeting. Because the largest subset of sensory neurons uses alpha- galactose-specific recognition during its target assembly, we will test for its possible binding to LL35, an endogeneous galectin with strong alpha-selectivity, using perturbations with Fab fragments and purified galectin. In order to determine whether the target assembly of these axons involves their interaction with postsynaptic neurons, we have developed ultrastructural procedures in which we can antibody-label live synaptic endings in order to preserve ultrastructural detail. Because antibody is taken up into cisternae, this ultrastructural procedure lends itself as an exciting strategy to use antibody staining as an activity- dependent marker. This will permit us to determine whether neuronal function is encoded by carbohydrate markers and therefore can nerve as suitable cell-cell recognition molecules during the formation of precise synaptic connectivity.
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