Using the MDCK organoid system, we found the following. 1) The fluorescent protein with the focal adhesion targeting (FAT) signal from the tyrosine kinase FAK, is localized to the basal membrane in MDCK mature cysts. Using this FAT signal, we are now constructing the plasmids of various proteins which are known to regulate the cytoskeleton. After establishing cell lines expressing these proteins, we will observe the localization of these tagged proteins in the organoid, and see if they induce malignant invasive morphological changes. 2) Expression of activated Ras in mature cysts accelerated cell cycle progression, leading to the luminal cell filling, which resembles adenoma or hyperplasia in vivo. We also found that the mature MDCK cysts coherently rotated upon the active Ras expression. To investigate the signaling pathways for this phenotype, mRNA expression with or without active Ras expression was examined by microarray. 3) By comparing the RNA expression at early and late phases of MDCK cystogenesis, we identified genes upregulated at the late phase. We examined one of these genes, Ripply1, and found that its mutant expression induced organoid “fission”, where cells move toward matrix (unpublished data).