Building the Cell\u27s Antenna: Protein Targeting to the Ciliary Membrane: A Dissertation

Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell\u27s ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane

Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease

Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation

Hypomorphic mutations of TRIP11 cause odontochondrodysplasia

Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism. In spite of reduced abundance, residual GMAP variants maintain partial Golgi integrity, normal global protein secretion, and subcellular distribution of IFT20 in ODCD. These functions are lost when GMAP-210 is completely abrogated in ACG1A. However, a similar defect in chondrocyte maturation is observed in both disorders, which produces a cellular achondrogenesis phenotype of different severity, ensuing from aberrant glycan processing and impaired extracellular matrix proteoglycan secretion by the Golgi apparatus

Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of ∼16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum

Arf4 is required for Mammalian development but dispensable for ciliary assembly

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes

The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence

An 18-residue motif in the cytoplasmic tail of polycystic kidney disease gene product, fibrocystin, targets it to ciliary membranes through interactions with Rab8

The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane

Induction of Ran GTP drives ciliogenesis

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin–Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport

Characterization of mouse IFT complex B

The primary cilium plays a key role in the development of mammals and in the maintenance of health. Primary cilia are assembled and maintained by the process of intraflagellar transport (IFT). In this work, we characterize mouse IFT complex B by identifying all of the mammalian orthologues of complex B and B-associated proteins previously identified in Chlamydomonas and Caenorhabditis and also identify a new component (IFT25/Hspb11) of complex B by database analysis. We tagged each of these proteins with the FLAG epitope and show that all except IFT172 and IFT20 localize to cilia and the peri-basal body or centrosomal region at the base of cilia. All of the proteins except IFT172 immunoprecipitate IFT88 indicating that they are co-assembled into a complex. IFT20 is the only complex B protein that localizes to the Golgi apparatus. However, overexpression of IFT54/Traf3ip1, the mouse orthologue of Dyf-11/Elipsa, displaces IFT20 from the Golgi apparatus. IFT54 does not localize to the Golgi complex nor does it interact with GMAP210, which is the protein that anchors IFT20 to the Golgi apparatus. This suggests that IFT54s effect on IFT20 is a dominant negative phenotype caused by its overexpression. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc