Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

BACKGROUND: The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional co-factors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIMD1 and WTIP) can bind and inactivate LATS1/2, two of the Hippo pathway key kinases. WTIP furthermore connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton.

METHODS: We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS regarding expression, localization, interaction, and effect on the functionality of the SD.

RESULTS: In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced Djub level. Both Djub knockdown and Hippo activation led to morphological changes of the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins.

CONCLUSIONS: Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.