Reversion von terminaler Differenzierung und Zellproliferationskontrolle

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The goal of this proposal is the elucidation of regulatory mechanisms controlling cell cycle reentry in terminally differentiated muscle cells. We have etablished a cell-free replication system with primary cardiomyocytes and were able to detect in extracts from adult cardiomyocytes an activity inhibiting S-phase entry. We will now use this cell free system to identify this inhibitor by immunodepletion of specific candidates or by biochemical fractionation techniques. Recently, we have shown that proteins can be directly delivered to differentiated muscle cells by fusion to the viral VP22 factor. We will now test whether VP22-SV40 Tag can induce both, S-phase and mitosis, in myotubes and cardiomyocytes. The use of Tag mutants combined with an analysis of effects on known cell cycle factors should allow us to identify the key regulators preventing cell cycle progression in differentiated muscle cells. Our studies on the dynamics of DNA replication foci and the subnuclear localization of cyclin A and cdk2 suggested that they are involved in the activation of DNA replication system we will now directly add fluorescently labelled cdk2 and cyclin A to screen for association with replication foci. We will then add cyclinA/cdk2 mutants to map binding sites and test their role in S-phase progression.