The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxidegenerating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1 Ϫ/Y ), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed.

A primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub-compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the "Inv compartment", which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra-ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra-ciliary interaction of Inv, Nphp3 and Nek8.

a b s t r a c tGlis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3 À/À mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulinproducing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.

Primary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1-11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled-coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences (CTSs) are unknown. In the present study, we generated a series of GFP-tagged deletion constructs of Nphp3 and tried to find the CTSs of Nphp3. We found that the N-terminal 201 amino acid fragment (Nphp3 [1-201]), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N-terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N-terminal fragments, Nphp3 (8-201), Nphp3 (52-201), and Nphp3 (96-201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two-step process.