Cilia serve as sensory devices on most eukaryotic cell surfaces and play essential roles in organogenesis and tissue pattern formation during 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 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 kidneys, liver limbs, eyes, central nervous system (CNS), and fat storage tissue. 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 poorly understood. Enzymatic small GTPases act as molecular switches, which control fundamental cellular processes and are often correlated with various human pathological conditions. Studies from other and our laboratories demonstrated that three conserved and poorly characterized ADP-ribosylation factor-like (ARL) small GTPases, ARL3, ARL6, and ARL13B, act as prominent ciliary switches, with disrupted function predisposing human or mice to ciliopathies. The paramount obstacle being that the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that switch ciliary ARLs on and off, respectively, and the effectors of ARLs have not been identified. In other disorders, such as tuberous sclerosis, identifying the GTPase inhibited by the TSC2 GAP was transformative in understanding the disease, and we propose that the corresponding knowledge here would have a similar dramatic effect on understanding ciliopathies. Due to highly conserved cilia pathways and ciliopathy genes, Caenorhabditis elegans has been established as a simple and effective model for characterizing the physiological roles of ciliopathy proteins in their native cellular environments. In last fundig period, we have successfully established C. elegans as a model to investigate the roles of ciliopathy ARLs. Our recent data suggested that ciliary ARLs are likely organized into two distinct functional modules in the enigmatic inversin (InV) compartment of cilia. One function module contains ARL-13-ARL-3-NPHP-2-UNC-119, in which UNC-119 and nephronophthisis protein NPHP-2 act synergistically with ARL-13, but antagonistically with ARL-3, in regulating ciliogenesis. The second one contains ARL-6-ARL-13-BBSome, which may regulate cilia signaling through regulating the proper localization of ciliary sensory receptors. Our preliminary results also supported that the roles of ciliopathy ARLs are highly conserved from worm to mammalian cells. Based on these, our central hypothesis is that the three ciliopathy ARLs and their regulators are organized into distinct complexes to coordinate cilia biogenesis and signaling, respectively. We will employ C. elegans to identify in vivo regulators and functions, and mammalian systems to determine the applicability to human ciliopathies.
Specific Aim 1 is to characterized the type of regulators for each component in ARL-containing protein module, and we hope to identify GEFs, GAPs, or effectors for ciliopathy ARLs;
Specific Aim 2 is to ascertain whether and how ARL-13, NPHP-2, and UNC-119 coordinate IFT integrity and/or axonemal stability in the InV compartment, and whether ARL-3 antagonizes the roles of ARL-13-NPHP-22-UNC-119 through deacetylase HDAC-6-dependent manner;
Specific Aim 3 is to determine whether the ARL-6-ARL-13-BBSome module coordinates cilia signaling through the mechanism that ARL-13 promotes ARL-6 activation, BBSome-cargo assembly, and subsequent proper ciliary localization of sensory receptors in the InV compartment. The proposed studies have great potentials for unveiling breakthroughs in cilia biology, 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, and potentially provide novel targets fo disease diagnosis and treatment.

Public Health Relevance

Defects in cilia biogenesis or function contribute to a wide spectrum of human genetic diseases, now collectively termed as ciliopathies. Small GTPases act as molecular switches, which control fundamental cellular processes and are often correlated with various human pathological conditions. This proposal is designed to combine the simple and effective genetic model C. elegans with cultured mammalian cells to mechanistically characterize the concerted and conserved roles of three poorly understood small GTPases (ARL3, ARL6, and ARL13B) in the context of cilia and their correlation to 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 the roles of the three ciliary ARLs and their regulators in disease processes, and their potential as therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK090038-08
Application #
9407307
Study Section
Kidney Molecular Biology and Genitourinary Organ Development (KMBD)
Program Officer
Maric-Bilkan, Christine
Project Start
2016-01-15
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
He, Kai; Ma, Xiaoyu; Xu, Tao et al. (2018) Axoneme polyglutamylation regulated by Joubert syndrome protein ARL13B controls ciliary targeting of signaling molecules. Nat Commun 9:3310
Chen, Chunhua; Wang, Xiangling; Fang, Juemin et al. (2017) EGFR-induced phosphorylation of type I? phosphatidylinositol phosphate kinase promotes pancreatic cancer progression. Oncotarget 8:42621-42637
Zhang, Qing; Li, Yan; Zhang, Yuxia et al. (2016) GTP-binding of ARL-3 is activated by ARL-13 as a GEF and stabilized by UNC-119. Sci Rep 6:24534
Xu, Qingwen; Zhang, Yuxia; Wei, Qing et al. (2016) Phosphatidylinositol phosphate kinase PIPKI? and phosphatase INPP5E coordinate initiation of ciliogenesis. Nat Commun 7:10777
Wei, Qing; Zhang, Yingyi; Schouteden, Clementine et al. (2016) The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate. Nat Commun 7:12437
Xu, Qingwen; Zhang, Yuxia; Wei, Qing et al. (2015) BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors. Sci Rep 5:11855
Wei, Qing; Ling, Kun; Hu, Jinghua (2015) The essential roles of transition fibers in the context of cilia. Curr Opin Cell Biol 35:98-105
Chen, C; Wang, X; Xiong, X et al. (2015) Targeting type I? phosphatidylinositol phosphate kinase inhibits breast cancer metastasis. Oncogene 34:4635-46
Xu, Qingwen; Zhang, Yuxia; Xiong, Xunhao et al. (2014) PIPKI? targets to the centrosome and restrains centriole duplication. J Cell Sci 127:1293-305
Zhang, Qing; Hu, Jinghua; Ling, Kun (2013) Molecular views of Arf-like small GTPases in cilia and ciliopathies. Exp Cell Res 319:2316-22

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