Gray RS , Abitua PB , Wlodarczyk BJ , Szabo-Rogers HL , Blanchard O , Lee I , Weiss GS , Marcotte EM , Wallingford JB , Finnell RH .

	Figure 2. RSG1 controls ciliogenesis and secretion. (a) SEM of intact, control Xenopus ciliated epidermis reveals multi-ciliated cells and surrounding mucous secreting cells (b) RSG1 morphants display defects in ciliogenesis and absence of mucous granules and exocytic pits. (c) Higher-magnification view of RSG1 morphant ciliated epidermis displaying diminished cilia numbers and lengths and a decrease in exocytic pits in neighboring secretory cells. (d) Fuz morphants also display diminished cilia numbers and lengths and a decrease in exocytic pits in secretory cells. (e, f) Epidermal targeted over-expression of RSG1T65N, but not wild type RSG1, results in defects in ciliogenesis as well as decreases in mucous granules and exocytic pits in secretory cells. (g, h) GFP-RSG1 (low-level expression) localizes to the basal body region of multi-ciliated cells, whereas GFP-RSG1T65N (low-level expression) in multi-ciliated cells is diffuse and not tightly associated with basal bodies. Observations of fluorescence levels following expression of GFP-RSG1 and GFP-RSG1T65N mRNA were comparable, suggesting similar rates of translation for the two proteins.
	Figure 3. Homology modeling, network predictions, and experimental validation suggest a trafficking function for Fuz. (a, b) Rendered protein models (Open-Source PyMOL 0.99rc6 software). (a) Sedl N-terminal domain (pdb:1H3Q). (b) 1H3Q based homology threaded model of the C- terminal -aa(s) 287–419 - of Xenopus Fuz protein. (c) Illustration of experimentally-derived protein-protein interactions (see Supplemental Methods) linking Dvl2 with longin-domain proteins AP1s, AP2s, and SedlP as well as CLAMP. (d) Mosaic imaging of live embryo expressing CLAMP-GFP, which localizes to ciliary axonemes and apical tips. A nucRFP marks the nuclei, serving as a lineage tracer for co-injected Fuz MO. The confocal slice reveals a loss of apical localized CLAMP-GFP at ciliary tips in FUZMO cells (inset shows merge of (e) and a more basal slice to display nucRFP signal). Scale bar in (e) = 10uM.
	Figure 4. Fuzzy and RSG1 control trafficking to basal bodies as well as to the tips of cilia. (a, b) Fuz knockdown disrupts localization of CLAMP-GFP to the ciliary rootlet. Confocal stacks of formaldehyde fixed Xenopus epidermis expressing centrin-RFP and CLAMP-GFP mRNAs. (a) Multi-ciliated cell in x-y view from an uninjected control embryo exhibits elongated CLAMP-GFP signal extending from relatively evenly spaced basal bodies (centrin-RFP). (a′) Higher magnification view of the x-y section from [a]. (a″) X-Z projection of the stack shown in [a′] displays apical co-localization of centrin-RFP and CLAMP-GFP. (b) Ciliated cell in an x-y view of the apical surface in a Fuz morphant reveals defects in the spacing of centrin-RFP signal and defects in elongated CLAMP-GFP signal. Additionally CLAMP-GFP signal is not faithfully co-localized with centrin-RFP signal in many cases. (b′) Higher magnification view of the x-y section from [b]. (b″) X-Z-projection of the stack shown in [b′] reveals apical alignment of the centrin-RFP signal however in many cases the CLAMP-GFP signal is below the apical surface in large punctae. (c–e) Mosaic imaging of live agarose embedded embryo. CLAMP-GFP highlights a variety of epidermal structures. RFP-Histone 2B (nuc-RFP) serves as a lineage tracer for morpholino-injected cells. (c, d) 3D projections (x-z). RSG1 MO (+ nucRFP cells) display defects in normal CLAMP-GFP localization along the apical surface. (e) Confocal slice (x-y) exhibiting a loss of apical localized CLAMP-GFP to ciliary tips in RSG1 MO cells (inset shows merge of (e) and more basal slice to display nucRFP signal). Scale bars in (c, d) are 3uM; scale bar in (e) is 10μm.
	Figure 5. Knockdown of Fuz disrupts exocytosis in mucus-secreting cells. (a) Wild type multi-ciliated cell (left) flanked by secretory cells in Xenopus mucociliary epidermis. Control cells have an average of over 90 open exocytic pits per cell. (b) Fuz morphants display defects in ciliogenesis in multi-ciliated cell (left) and failure of exocytosis in mucus-secreting cells (note the absence of exocytic pits indicated by white arrowheads in [a]). Green arrowheads indicate apical membrane blebs (see also panel F, below). In a representative experiment, Fuz morphant cells had fewer than 5 exocytc pits per cell on average (difference from control is significant by the Mann-Whitney U-test; p<0.0001). Scale bars in A, B = 5μm (c) Mosaic epidermal tissue, with morphant cells outlined in red. Scale bar = 10μm (d) Confocal section (x-y) of mosaic embryo in which membrane-RFP (memRFP, red) mRNA was co-injected with Fuz morpholino and processed for Xeel (interlectin-2) antibody (green). Cell expressing a high level memRFP (right) lacks apical Xeel antibody staining comparable to the neighboring cell (right). (d′) Confocal projection (x-z) of [d] illustrates the loss of apical Xeel staining (green) in the Fuz morphant cell correlated with apical memRFP expression (right). Scale bars in D, d′ = 3μm (e) TEM of section of control Xenopus epidermis shows empty and mucus granule-containing vesicles docked at the apical surface, whereas Fuz morphants (f) display a defect of vesicle fusion with the plasma membrane release, as illustrated by large membrane protrusions (green arrows in [b, f]). Additionally, frequent homotypic vesicle fusion events were observed in secretory cells of Fuz morphants (red arrow). Scale bar in E = 500nm; Scale bar in F= 100nm.

Ansley, Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome. 2003, Pubmed

Ansley, Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome. 2003, Pubmed
Beales, IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy. 2007, Pubmed
Billett, Fine structural changes in the differentiating epidermis of Xenopus laevis embryos. 1971, Pubmed , Xenbase
Boyadjiev, Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking. 2006, Pubmed
Cai, Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. 2007, Pubmed
Chan, Spef1, a conserved novel testis protein found in mouse sperm flagella. 2005, Pubmed
Collier, Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein. 1997, Pubmed
Collins, Molecular architecture and functional model of the endocytic AP2 complex. 2002, Pubmed
Dougherty, CLAMP, a novel microtubule-associated protein with EB-type calponin homology. 2005, Pubmed
Fariss, Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways. 1997, Pubmed
Follit, The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. 2006, Pubmed
Gedeon, Identification of the gene (SEDL) causing X-linked spondyloepiphyseal dysplasia tarda. 1999, Pubmed
Ginalski, Comparative modeling for protein structure prediction. 2006, Pubmed
Hagiwara, Development of striated rootlets during ciliogenesis in the human oviduct epithelium. 1997, Pubmed
Hamblet, Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure. 2002, Pubmed
Hayes, Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development. 2007, Pubmed , Xenbase
Huangfu, Hedgehog signalling in the mouse requires intraflagellar transport proteins. 2003, Pubmed
Jang, Crystal structure of SEDL and its implications for a genetic disease spondyloepiphyseal dysplasia tarda. 2002, Pubmed
Karner, Planar cell polarity and vertebrate organogenesis. 2006, Pubmed
Kibar, Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail. 2001, Pubmed
Lang, Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation. 2006, Pubmed
Lee, The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy. 2002, Pubmed
Nachury, A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. 2007, Pubmed
Nagata, Developmental expression of XEEL, a novel molecule of the Xenopus oocyte cortical granule lectin family. 2003, Pubmed , Xenbase
Park, Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. 2008, Pubmed , Xenbase
Park, Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. 2006, Pubmed , Xenbase
Peña-Castillo, A critical assessment of Mus musculus gene function prediction using integrated genomic evidence. 2008, Pubmed
Pryor, Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb. 2008, Pubmed
Ross, Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. 2005, Pubmed
Rossi, Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators. 2004, Pubmed
Rual, Towards a proteome-scale map of the human protein-protein interaction network. 2005, Pubmed
Sharma, Ciliary dysfunction in developmental abnormalities and diseases. 2008, Pubmed
Simons, Planar cell polarity signaling: from fly development to human disease. 2008, Pubmed
Smith, The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat. 2006, Pubmed
Sorokin, Reconstructions of centriole formation and ciliogenesis in mammalian lungs. 1968, Pubmed
Takeda, Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis. 1999, Pubmed
Wallingford, Planar cell polarity, ciliogenesis and neural tube defects. 2006, Pubmed
Yang, The ciliary rootlet interacts with kinesin light chains and may provide a scaffold for kinesin-1 vesicular cargos. 2005, Pubmed