Renal-retinal ciliopathy gene Sdccag8 regulates DNA damage response signaling.

Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8(gt/gt) mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to progressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8(gt/gt)-derived cells, characterized by elevated levels of γH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8(gt/gt) mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specific disease mechanisms.

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms.

The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1-EB3 dimer species have distinct functions within cells.

EB1 is required for primary cilia assembly in fibroblasts.

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles [1, 2]. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network [3, 4]. We previously showed that EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas[5], but the function of EB1 in the cilium/flagellum is unknown. We depleted EB1 from NIH3T3 fibroblasts by using siRNA and found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibited cilia formation, and expression of mutant dominant-negative EB1 constructs suggested that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends [6], also inhibited ciliogenesis. We conclude that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-ß/BMP Signaling at the Primary Cilium.

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.

SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.

The ciliary cytoskeleton.

Cilia and flagella are surface-exposed, finger-like organelles whose core consists of a microtubule (MT)-based axoneme that grows from a modified centriole, the basal body. Cilia are found on the surface of many eukaryotic cells and play important roles in cell motility and in coordinating a variety of signaling pathways during growth, development, and tissue homeostasis. Defective cilia have been linked to a number of developmental disorders and diseases, collectively called ciliopathies. Cilia are dynamic organelles that assemble and disassemble in tight coordination with the cell cycle. In most cells, cilia are assembled during growth arrest in a multistep process involving interaction of vesicles with appendages present on the distal end of mature centrioles, and addition of tubulin and other building blocks to the distal tip of the basal body and growing axoneme; these building blocks are sorted through a region at the cilium base known as the ciliary necklace, and then transported via intraflagellar transport (IFT) along the axoneme toward the tip for assembly. After assembly, the cilium frequently continues to turn over and incorporate tubulin at its distal end in an IFT-dependent manner. Prior to cell division, the cilia are usually resorbed to liberate centrosomes for mitotic spindle pole formation. Here, we present an overview of the main cytoskeletal structures associated with cilia and centrioles with emphasis on the MT-associated appendages, fibers, and filaments at the cilium base and tip. The composition and possible functions of these structures are discussed in relation to cilia assembly, disassembly, and length regulation.

Assembly of primary cilia.

Primary cilia are microtubule-based, hair-like sensory organelles present on the surface of most growth-arrested cells in our body. Recent research has demonstrated a crucial role for primary cilia in regulating vertebrate developmental pathways and tissue homeostasis, and defects in genes involved in primary cilia assembly or function have been associated with a panoply of disorders and diseases, including polycystic kidney disease, left-right asymmetry defects, hydrocephalus, and Bardet Biedl Syndrome. Here we provide an up-to-date review focused on the molecular mechanisms involved in the assembly of primary cilia in vertebrate cells. We present an overview of the early stages of the cilia assembly process, as well as a description of the intraflagellar transport (IFT) system. IFT is a highly conserved process required for assembly of almost all eukaryotic cilia and flagella, and much of our current knowledge about IFT is based on studies performed in Chlamydomonas and Caenorhabditis elegans. Therefore, our review of the IFT literature includes studies performed in these two model organisms. The role of several non-IFT proteins (e.g., centrosomal proteins) in the ciliary assembly process is also discussed.