Molecular and cellular characterization of Myosin-V in brain and cultured cells: function and regulation

Abstract

The myosin superfamily includes about 13 classes of structurally distinct molecules. While the discovery of new classes of myosin is becoming more remote, new members of existing classes are still being identified in many organisms and cell types. The present challenge is to define the exact cellular role of each of these myosins. We are investigating the biochemical, structural and cellular properties of myosin-V, a class of unconventional myosins featuring an extended neck domain consisting of 6 tandem repeats of the IQ-motif, potential binding sites for calmodulin and other light chains, and a structurally distinct tail domain which we suppose to be involved in cellular localization, anchoring and/or other specific functions of this myosin. We are following 3 general approaches: 1) The structural and biochemical characterization of the native protein and its isolated or bacterially expressed domains. The major questions are: why does myosin-V have such a large extended neck domain? and what are the properties and function of its tail domain? 2) The transformation of eukaryotic cells in culture (melanocytes and PC12 cells) by cDNA corresponding to myosin-V and its specific domains. We are applying video-microscopy techniques with the Metamorph softeware analysis system and confocal microscopy to analyse the phenotypes of the transformants and the exact subcellular localization of myosin-V. 3) Immunolocalization and analysis of expression of myosin-V during neural ontogenesis and in experimental models of epilepsy. We are applying immunolocalization and in situ hybridization techniques to investigate correlations between the localization/expression of myosin-V and neurological events associated with development, synaptogenesis and induced convulsions in experimental animals. (AU)