Primary cilia are dynamic, complex structures that contain >250 proteins, including several polycystic kidney disease (PKD)-related proteins. In renal epithelial cells, the primary apical cilium appears to be a major effector of differentiation signals and to play a critical role in PKD pathogenesis. Recent in vitro studies demonstrate that the primary cilium acts as a cellular sensor, transducing apical mechanical signals through a polycystin-1/polycystin-2-dependent Ca++ signaling pathway. However, the precise mechanisms involved in cilia formation, stabilization, and signal transduction are not well-defined and even less is known about how these cilia-associated proteins are targeted to cilia and functionally assembled. We have identified Cys1 as the disease-gene in cpk mice; demonstrated that its novel protein product, cystin, localizes to the primary apical cilium; and determined that cystin fractionates with lipid rafts through an N-terminal domain, probably the predicted N-myristoylation/ polybasic motif. We hypothesize that cystin traffics to the primary cilium via lipid raft-mediated mechanisms, associates with the cilial membrane, and serves as part of the molecular framework that stabilizes the microtubular scaffold of the ciliary axoneme. Using a suite of stably transfected cell lines that express wild-type cystin and various truncation mutants as GFP-tagged fusion proteins, we have determined that the N-terminal domain is necessary but not sufficient for targeting cystin to cilia and a second, novel signal is required. Since cystin is expressed at low levels and no functional assays currently exist, we have developed an innovative set of strategies to further characterize this novel protein and its intracellular trafficking itinerary as first steps toward defining its function. Specifically, in this proposal, we will: 1) Determine whether cystin tagged with green fluorescent protein (cystin-GFP) rescues the cpk phenotype and targets correctly to the primary cilium of renal epithelia in vivo; 2) Characterize cystin with respect to the predicted N-myristoylation site, putative cilia-targeting signals, and putative interacting partners; and 3) Examine the dynamics of cystin intracellular trafficking to the primary apical cilium. The central hypotheses underlying the proposed studies are that defects in primary cilia function impair the terminal phases of renal tubulo-epithelial differentiation and the epithelial response to this developmental arrest is cyst formation. Therefore, primary apical cilium represents a new focal point for dissecting the complex mechanisms involved in renal cystic disease and ultimately, perhaps a new target for therapeutic interventions. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK055534-05
Application #
6826566
Study Section
Special Emphasis Panel (ZRG1-RUS-D (02))
Program Officer
Rasooly, Rebekah S
Project Start
2000-03-15
Project End
2009-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
5
Fiscal Year
2004
Total Cost
$333,199
Indirect Cost
Name
University of Alabama Birmingham
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Wu, Maoqing; Yang, Chaozhe; Tao, Binli et al. (2013) The ciliary protein cystin forms a regulatory complex with necdin to modulate Myc expression. PLoS One 8:e83062
Raynaud, Peggy; Tate, Joshua; Callens, CĂ©line et al. (2011) A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis. Hepatology 53:1959-66
Steele, Stacy L; Wu, Yongren; Kolb, Robert J et al. (2010) Telomerase immortalization of principal cells from mouse collecting duct. Am J Physiol Renal Physiol 299:F1507-14
Tao, Binli; Bu, Su; Yang, Zhihua et al. (2009) Cystin localizes to primary cilia via membrane microdomains and a targeting motif. J Am Soc Nephrol 20:2570-80
Mrug, M; Zhou, J; Woo, Y et al. (2008) Overexpression of innate immune response genes in a model of recessive polycystic kidney disease. Kidney Int 73:63-76
Li, Jin Billy; Gerdes, Jantje M; Haycraft, Courtney J et al. (2004) Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117:541-52
Guay-Woodford, Lisa M (2003) Murine models of polycystic kidney disease: molecular and therapeutic insights. Am J Physiol Renal Physiol 285:F1034-49
Hou, Xiaoying; Mrug, Michal; Yoder, Bradley K et al. (2002) Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J Clin Invest 109:533-40
Mrug, M; Green, W J; DasGupta, S et al. (2001) An integrated genetic and physical map of the 650-kb region containing the congenital polycystic kidney (cpk) locus on mouse chromosome 12. Cytogenet Cell Genet 94:55-61