Influence of Rho GTPases on the regulation of neuronal dendritic morphology and the establishment of neuronal polarity

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Rho GTPases are molecular switches that regulate actin polymerisation in all eukaryotic cells (Hall, 1998). Recent work has suggested that Rho GTPases regulate processes in neurons leading to changes in spine and dendritic morphology (Luo et al., 1996) and to the establishment of neuronal polarity (Bradke and Dotti, 1999). The aim of my proposal is to characterize the molecular role of Rho GTPases in these processes. I want to investigate two aspects: A) Regulation of the rapid changes in motility and morphology of mature dendritic spines, and B) Regulation of dendritic/axonal differentiation, a slower process involving cell polarization, actin and microtubule (MT) dynamics and membrane transport. For A) I want to establish techniques, by which I can interfere with endogenous Rho GTPase activity rapidly and locally restricted in order to mimic physiological conditions of Rho GTPase activation. This will be achieved by using local superfusion techniques of clostidium toxins or cell permeable Rho GTPase proteins or by local uncaging of caged Rho GTPase mutants. Neuronal morphology will be imaged at the same time using fluorescently tagged membrane or cytoskeletal proteins. For B) a culture system will be established, suitable für studying the initial establishment of neuronal polarity and for influencing Rho GTPases activity during all stages of neuronal polarisation.