Suzanne de Bruijn

110 Chapter 3.1 cells ( Figure 5A ). Morphology of the stereocilia was not significantly affected two days after injectoporation of the mutant Ripor2 construct, suggesting the mutant protein did not visibly affect the stereocilia structure in the short term. A HA-RIPOR2wt HA-RIPOR2wt/Phal HA-RIPOR2mut HA-RIPOR2mut/Phal Phal Phal/HA-RIPOR2wt Phal Phal/HA-RIPOR2mut B Figure 5. Functionality of mutant RIPOR2 is altered in mouse cochlear outer hair cells. A. Mutant RIPOR2 differed in localization from wildtype RIPOR2 in mouse cochlear outer hair cells. Outer hair cells of wildtype mice were injectoporated at P2 to express murine N-terminally HA-tagged wildtype RIPOR2 (RIPOR2wt) or mutant RIPOR2 (RIPOR2mut). Expression was evaluated after two days by immunohistochemistry and three representative images of cells expressing the mutant RIPOR2 are provided. Eleven cells expressing the wildtype construct and 12 cells expressing the mutant construct were evaluated. B. Mutant RIPOR2 did not rescue stereocilia defects in RIPOR2-deficient hair cells. Cochlear explants of RIPOR2-deficient mice were prepared at P2 and injectoporated with constructs RIPOR2wt or RIPOR2mut. After culturing for two days, five out of six cells expressing the wildtype RIPOR2 construct demonstrated rescued hair bundle morphology but none of the 13 cells expressing the mutant RIPOR2 construct. Cells expressing the constructs are boxed. HA-tagged protein was stained in green, stereocilia were stained using phalloidin (phal) conjugated with Alex Fluor 568 (red). Scale bar represents 5 µm. Potentially, the variant affects interactions of RIPOR2 that are essential for its localization. The four deleted amino acids are predicted to be part of a disorganized coiled coil structure (predicted using KMAD 25 ). Coiled coil regions are indicated tomediate protein- protein interactions, which supports the hypothesis that the variant affects RIPOR2 protein interactions. 26 Co-immunoprecipitation (Co-IP) assays demonstrated that both the dimerization ability ( Figure S8A ) and the interaction with RHOC ( Figure S8B ) of mutant RIPOR2 are intact.