Processing of the human protocadherin Fat1 and translocation of its cytoplasmic domain to the nucleus.

The giant protein hFat1, a member of the cadherin superfamily, has been proposed to play roles in cerebral development, glomerular slit formation, and also to act as a tumor suppressor, but its mechanisms of action have not been elucidated. To examine functions of the transmembrane and cytoplasmic domains, they were expressed in HEK293 and HeLa cells as chimeric proteins in fusion with EGFP and extracellular domains derived from E-cadherin. Proteins comprising the transmembrane domain localized to the membrane fraction. Deletion of this domain resulted in a predominantly nuclear localization of the cytoplasmic segment of hFat1. Nuclear localization was largely reduced by deletion of a presumed juxta-membrane NLS. Fusion proteins located in the plasma membrane underwent proteolytic processing. In a first proteolytic step, only the extracellular domain was cleaved off. In another step, the cleavage product was released to the cytosol and was also found in a low speed pellet fraction, in accordance with the nuclear localization of the cytoplasmic domain of hFat1.

[Liver organ systems: valuation by gene expression]

The multitude of vital functions of the liver is largely dependent on the complex interaction of hepatocytes with "littoral-cells" and the liver-specific extracellular matrix. In vitro systems used to study the liver are almost exclusively based on the culture of single cells obtained by enzymatic disruption of the essentiel organ structure. In contrast, the slicing technique enables a standardized organ culture, while retaining the intact organ structure. However, previous experiments using this system have been restricted to short-term pharmacological studies and have used general cellular parameters to assess viability. The lack of liver specific validation has hindered the acceptance and application of this organ culture as an alternative to in vivo experiments. In this work we chose the regulation of liver-specific gene expression as an adequate viability criterion because, in contrast to the commonly used general cell physiology parameters, gene expression includes a cascade of differentially coordinated processes. Using hormonal responsiveness of precision-cut liver slices as a basis for comparison, the new developed interphase system was found to be superior to a perfusion system. With the application of this economical and easy-to-handle interphase system, the control of liver-specific gene expression by dexamethasone, cAMP and endotoxin in long-term cultures was found to be modulated in a manner similar to in vivo conditions.