Primary cilia protrude from most mammalian cells and modulate sensory processes, including chemo-, mechano-, and photo-reception (Fliegauf et al., 2007
). Cilia regulate various signaling pathways during embryonic development and are needed for normal postnatal tissue homeostasis (Gerdes et al., 2009
). Mutations disrupting ciliary functions cause human disorders (ciliopathies) that collectively affect nearly all tissues/organs (Sharma et al., 2008
). A nonexhaustive list of ciliopathies includes Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS), Senior-Løken syndrome (SLSN), Leber congenital amaurosis (LCA), polycystic kidney disease (PKD), and oral-facial-digital syndrome (OFD). These disorders present with variable but overlapping clinical phenotypes that encompass polycystic kidneys, liver fibrosis, skeletal anomalies, sensory impairment, and brain/nervous system deformities (Fliegauf et al., 2007
At least 35 loci have been identified in ciliopathy patients, some of which contribute to multiple seemingly distinct syndromes (Baker and Beales, 2009
). Many of these genes encode proteins that localize to the basal body (BB)—a centriolar structure universally required for extending the microtubule-based ciliary axoneme—or to an adjacent domain, termed “transition zone” (TZ) in most cilia, or “connecting cilium” in photoreceptors (Horst et al., 1990
; see schematic of BB-TZ-cilia structures in and relevant disease proteins in Table S1 A
). Within the BB-TZ region are subdomains that include transitional fibers (TFs) and Y-links. TFs form a pinwheel-like structure, of unknown protein composition, that links the BB to the proximal ciliary membrane. The Y-links of the TZ connect—via high-affinity linkages—axonemal microtubules to the membrane at the ciliary necklace, a proteinaceous decoration of the TZ membrane (Muresan and Besharse, 1994
). Together, the TFs and TZ are proposed to form a gate (Rosenbaum and Witman, 2002
; Satir and Christensen, 2007
) that excludes vesicles from cilia, prevents unwanted diffusion of membrane proteins into cilia, and selectively regulates protein ciliary entry and exit ().
Figure 1. C. elegans B9 and C2 domain ciliopathy proteins are found at the transition zone, adjacent to the basal body/transition fiber region. (A) Schematic of a prototypical basal body (BB)/transition zone (TZ)/cilium, highlighting the microtubule (MT) backbone (more ...)
Axoneme elongation is thought to initiate when the mother centriole docks with a membrane either at the cell surface or a ciliary vesicle in the cytosol (see ; Sorokin, 1962
). Full extension of the axoneme then relies on an intraflagellar transport (IFT) machinery that uses kinesin and dynein motors and associated subcomplexes (IFT-A, IFT-B, and BBSome) to traffic ciliary cargo from TFs to the cilium tip and back (; Silverman and Leroux, 2009
). In contrast to the extensive characterization of IFT-mediated axoneme extension, the components and mechanism involved in BB/TZ membrane association and establishment of the ciliary gate remain virtually unknown. Based on knockdown studies in mammalian cells, the ciliopathy proteins MKS1 and MKS3 have been implicated in BB migration/docking and thus ciliogenesis (Dawe et al., 2007
). However, these defects are absent from rodent Mks1
mutants (Tammachote et al., 2009
); thus, the role of these and most other BB/TZ-associated ciliopathy proteins remains unclear. Recently, the ciliopathy protein CEP290 was localized to TZ Y-links, and its disruption in Chlamydomonas
altered the ciliary composition of IFT components and other proteins (Craige et al., 2010
); Caenorhabditis elegans
NPHP-1 and NPHP-4 have also been proposed to act in ciliary gating (Jauregui et al., 2008
). Whether additional TZ or IFT proteins are similarly involved in regulating ciliary gating, and the mechanism by which they perform these functions, is not known.
Figure 10. MKS/MKSR and NPHP proteins form part of a functional interaction network required for an early stage of ciliogenesis and formation of an intact ciliary gate. (A) Previously identified physical interactions between C. elegans or mammalian TZ proteins (see (more ...)
Here, we used C. elegans to elucidate the functions of eight conserved proteins, six of which are MKS/NPHP associated. We find that MKS-5/RPGRIP1L interacts with two distinct TZ functional modules, MKS/MKSR and NPHP, consisting of MKS-1/MKSR-1/MKSR-2/MKS-3/MKS-6 and NPHP-1/NPHP-4 proteins, respectively. Functional interactions between different MKS module components and the NPHP module are essential for an IFT-independent early stage of ciliogenesis, namely docking/anchoring of the BB/TZ to the membrane. Moreover, the two modules restrict inappropriate accumulation of membrane-associated proteins inside cilia. Our findings help to comprehensively define an interaction network of ciliopathy-associated proteins and allow us to propose for the first time a unified model for the function of diverse MKS/NPHP proteins, in which the MKS and NPHP modules altogether enable associations between microtubules and the ciliary membrane; this ciliogenic event coincides with construction of the ciliary gate that establishes the specialized compartment.