Cilia serve as sensory devices on most eukaryotic cells surface and play an essential role in the proper formation of a diversity of organs in development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders, such as autosomal dominant polycystic kidney disease (ADPKD), Joubert syndrome (JBST), Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), and autosomal recessive polycystic kidney disease (ARPKD), have been characterized molecularly as cilia-related diseases, now known collectively as ciliopathies. The establishment and maintenance of ciliary function are clearly essential for the well-being of an organism. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidney, liver, limb, eye, and central nervous system. Despite the physiological and clinical relevance of cilia, the core machinery that regulates cilia biogenesis and function as well as the connection between the disease gene function and pathology remain largely elusive. One central question in cilia biology is that how the ciliary gat functionally separates the cilium from the cell body and makes it a discrete sensing organelle. Cilia only form atop mother centrioles (or basal bodies). During ciliogenesis, the distal appendages of the mother centriole transform to transition fibers (TFs), which form a 9-bladed propeller structure connecting the basal body to the ciliary base membrane. The distinct subcellular location of TFs makes it a good candidate for the ciliary gate. Nonetheless, no molecular information is available regarding the composition as well as the function of TFs. In a forward mutagenesis screening that aimed to identify the determinants of ciliogenesis in C. elegans, we isolated and cloned a novel gene dyf-19, which is the sole homolog of poorly characterized human fbf1 gene. Our preliminary data showed that worm DYF-19 and human FBF1 exhibit specific localization pattern on transition fibers and distal appendages, suggesting a highly conserved TF-related function for DYF- 19/FBF1. Further analyses suggested that DYF-19 regulates the ciliary entry of assembled IFT particles on transition fibers as well as the ciliary entry of several ciliary sensory receptors. Our preliminary studies reveal the first bona fde component of TFs and demonstrate the essential roles of the TFs in cilia formation and function. Additionally, we identified two more genuine TF components, TALPID-3 and HYLS-1. Our preliminary data indicate that DYF-19, TALPID-3, and HYLS-1 functionally interact in the context of TFs. Most interestingly, talpid-3 knockout chicken is confirmed to be a ciliopathy model and human hyls1 gene is one causal locus for the ciliopathy Hydrolethalus syndrome. Due to the essential roles of cilia in mammalian early embryonic development, the study of the connections between cilia and disease are extremely difficult in humans and other mammalian model organisms. Thus, alternative experimental systems are necessary. C. elegans enables the exploration of these questions in living animals. The highly conserved ciliogenic proteins, ciliogenesis pathway, and cilia sensory function make Caenorhabditis elegans a powerful model for characterizing the physiological roles of ciliary genes in their native cellular environments. Our data support the central hypothesis of this proposal that DYF-19 acts as a functional component to define TFs as a "ciliary gate" that governs access of nascent proteins into the cilia. and that disruption of this "gate" compromises cilia formation and function. The proposed studies have great potential to unveil breakthroughs in cilia research in the near future, and would provide seminal information about how cilia biogenesis and sensory function are regulated in their native environment, shed light on the etiologies of ciliopathies.

Public Health Relevance

Defects in cilia biogenesis or function contribute to a wide spectrum of human diseases, now collectively called as ciliopathies. One central question in the ciliary biology is that how the ciliary base functionally separates the cilium from the cell body and makes it a discrete sensing organelle. This proposal is designed to use the simple but powerful genetic model C. elegans to molecularly dissect the composition and function of the ciliary gate as well as the correlation between the ciliary gate and the pathology of human ciliopathies. Our proposed studies will broaden the understanding of cilia development and function in normal and pathological states and provide seminal insights into how the ciliary gate is involved in this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK099160-01A1
Application #
8690500
Study Section
Special Emphasis Panel (KMBD)
Program Officer
Rasooly, Rebekah S
Project Start
2014-08-01
Project End
2018-03-31
Budget Start
2014-08-01
Budget End
2015-03-31
Support Year
1
Fiscal Year
2014
Total Cost
$238,500
Indirect Cost
$88,500
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905