UCSF

Primary cilia are signaling organelles that project from the apical surface of almost all vertebrate cells. The signaling capabilities of primary cilia stem from their unique membrane protein composition: even though the ciliary membrane is continuous with the plasma membrane, certain membrane proteins localize specifically to the ciliary membrane. This dissertation describes how the ciliary membrane protein composition is maintained, and how the complex responsible for ciliary membrane composition is localized. Using a combination of molecular biology, cell biology, and biochemistry, I have investigated the role of TMEM231 in the formation and function of the transition zone in cilia during development and in disease, and the role of INTU in localizing TMEM231 during ciliogenesis.

I show that TMEM231 is a component of the Meckel Syndrome (MKS) complex, and is responsible for organizing the MKS complex at the transition zone. In the absence of Tmem231, mouse embryos display phenotypes similar to those found in MKS, which is a genetic disorder characterized by polydactyly, cystic kidneys, and exencephaly. Using co-immunoprecipitation, I confirmed that TMEM231 physically interacts with other known MKS complex components, including B9D1 and MKS1. In addition, I show that TMEM231 functions with the MKS complex, which is required for ciliary membrane proteins to localize properly. In collaboration with human geneticists, we identified compound heterozygous mutations in TMEM231 in Oro-facio-digital syndrome type III (OFD3) patients, as well as homozygous mutations in TMEM231 in MKS patients. I discovered that these mutations are hypomorphic, as they are unable to restore full ciliary membrane protein localization in a rescue assay. These findings confirm that TMEM231 is a functional component of the MKS complex, and suggest that the mutations identified in OFD3 and MKS patients underlie the disease phenotypes.

In addition, I investigated the role of INTU in cilia function, because Intu mutant mice display phenotypes similar to MKS complex mutant mice. First, I confirm that INTU is necessary for cilia formation, and that this is not due to loss of IFT-B components. I observed that MKS complex components, including TMEM231 and TMEM67, are lost in the absence of Intu. Interestingly, another complex called the Nephronophthisis (NPHP) complex is also mislocalized. These results suggest that Intu is required for both NPHP and MKS complexes to localize properly. Finally, I looked at RPGRIP1L, the previously identified lynchpin of the NPHP and MKS complexes, and found that RPGRIP1L localizes normally in the absence of Intu. These data suggest that INTU functions downstream of RPGRIP1L to localize both the NPHP and MKS complexes. Cumulatively, I have shown that TMEM231 and INTU are required for cilia to form and function properly, and that cilia are important for regulating vertebrate development and adult tissue homeostasis.