Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract??? Cilia play key roles in development and homeostasis, and defects in cilia structure or function lead to an array of human diseases. Ciliogenesis is accomplished by the intraflagellar transport (IFT) system, a set of proteins governing bidirectional transport of cargoes within ciliary axonemes. In this paper, we present a novel platform for in vivo analysis of vertebrate IFT dynamics. Using this platform, we show that the planar cell polarity (PCP) effector Fuz was required for normal IFT dynamics in vertebrate cilia, the first evidence directly linking PCP to the core machinery of ciliogenesis. Further, we show that Fuz played a specific role in trafficking of retrograde, but not anterograde, IFT proteins. These data place Fuz in the small group of known IFT effectors outside the core machinery and, additionally, identify Fuz as a novel cytoplasmic effector that differentiates between the retrograde and anterograde IFT complexes.
Figure 1. Fuz is essential for distal, but not proximal, axonemal identity. (a, left) A representative control axoneme expressing RFP-CLAMP (top) and pixel intensity along the axoneme (bottom). (right) A collection of individual control axonemal intensity plots. (b, left) A representative axoneme from a Fuz KD cell and its corresponding intensity plot. This image is magnified compared with the image in a to facilitate comparison. (right) A collection of intensity plots from individual Fuz KD axonemes. Arrowheads indicate ectopic CLAMP enrichments. (c) Cilia on a control vertebrate multiciliated cell expressing RFP-CLAMP to label the distal axoneme and GFP-MAP7 to label the proximal axoneme. (d) Cilia on a Fuz KD vertebrate multiciliated cell expressing RFP-CLAMP and GFP-MAP7. Also see Fig. S1 (a and b). The mean percentage of axoneme length ± SD occupied by the CLAMP or MAP7 domains is indicated on the bottom right of c (middle [n = 54] and right [n = 130]) and d (middle [n = 52] and right [n = 130]). See Fig. S1 (d and e) for quantitation; only axonemes whose whole length was obvious were used for this analysis, and the compartment analysis for this dataset is shown in Fig. S1 g. (aâd) Bars, 3 µm. (e) Schematic representations of control and Fuz KD axonemes (Axo) showing mean proximodistal organization (for quantification, see Fig. S1 [d, e, and g]). Note that in Fuz KD axonemes, the MAP7 domain is of approximately the same length but makes up a greater percentage of the overall shorter axoneme. Also, note the reduction in both length and percentage of occupancy of the CLAMP compartment. Lengths are reported as the mean across the population, and the percentage of occupancies is reported as the mean of ratios from individual axonemes and is therefore not directly comparable. See Materials and methods for details. (f) Fuz KD axonemes show reduced enrichment of RFP-CLAMP signal in the distal domain over basal axoneme levels, as compared with controls (Ctl). Enrichment Index = (mean intensity compartment)/(mean intensity of an equivalent length of nonenriched axoneme). Fuz KD enrichment index (mean ± SD = 2.17 ± 0.70; n = 113) versus control enrichment index (4.28 ± 1.68; n = 87; P < 0.0001) is shown. Note that only axonemes with discernable distal RFP-CLAMP compartments were used for this analysis. (g) There is no change in the enrichment of the MAP7 compartment between control and Fuz KD axonemes. Fuz KD enrichment index (mean ± SD = 2.64 ± 0.64; n = 102) versus control enrichment index (2.65 ± 0.80; n = 75; P = 0.6508) is shown.
Figure 2. In vivo imaging of IFT in Xenopus multiciliated cells. (a) Still frame from a video of GFP-IFT20 in a multiciliated cell (Video 1). The orange box indicates the region shown in the bottom image, depicting still frames from a time-lapse video showing processive bidirectional movement of IFT particles in a single control cilium (Video 2). Time is indicated in seconds. Pink arrowheads indicate anterograde particles; blue arrowheads indicate retrograde particles. The axoneme is outlined in yellow. Bars, 3 µm. (b) Histogram of anterograde rates of IFT in Xenopus multiciliated cells. (c) Histogram of retrograde rates. (c and d) Mean velocities ± SD are 0.84 ± 0.22 µm/s anterograde (n = 143 trains from 24 cells; six embryos) and 0.87 ± 0.22 µm/s retrograde (n = 125 trains from 25 cells; six embryos). Velocities ranged from 0.36 to 1.35 µm/s anterograde and 0.42 to 1.43 µm/s retrograde. Velocities were calculated as the mean of three independent instantaneous velocities for each reported particle.
Figure 3. Loss of Fuz leads to disrupted anterograde IFT. (a) Still frame from a video of GFP-IFT20 in a Fuz KD cell (Video 5). The orange box indicates the region shown in the bottom image, depicting still frames from a time-lapse video revealing abnormally large and immotile IFT particles (red arrowheads) in a Fuz KD axoneme. The final frame is taken from the end of the time series (Video 6). The axoneme is outlined in yellow. (b) In a fixed control embryo, IFT particles (green) can be seen at low density in the axonemes of a multiciliated cell (red). (c) In a mildly affected Fuz KD embryo, IFT particles accumulate at high density throughout the axonemes. (d) GFP-IFT20 particles are static for >4 min in Fuz KD axonemes. Red brackets indicate especially clear examples. Bars, 4 µm.
Figure 4. Fuz is required for the axonemal localization and dynamics of retrograde IFT. (a) Punctate localization of the IFT-A component GFP-IFT43 in control multiciliated cell axonemes (marked by memRFP). (b) Reduced GFP-IFT43 in the axonemes of Fuz KD multiciliated cells. (c) Still frame from a video of a control multiciliated cell expressing GFP-IFT43 (Video 9). The orange box indicates the region shown in the bottom image, depicting still frames from a video showing processive bidirectional transport in a single control cilium (Video 10). Time is indicated in seconds. Pink arrowheads indicate anterograde particles; blue arrowheads indicate retrograde particles. (d) A single frame from a time-lapse video of a Fuz KD multiciliated cell expressing GFP-IFT43. The image has been intentionally overexposed to bring out the faint background fluorescence of the axonemes. The orange box indicates the region shown in the bottom image, depicting still frames from a video showing loss of GFP-IFT43 dynamics in a single cilium from a Fuz KD multiciliated cell. The axoneme is outlined in yellow. Bars, 3 µm.
Figure 5. Fuz is required for the localization of anterograde but not retrograde IFT proteins to peri-basal body pools in the apical cytoplasm. (aâd) Pools of GFP-IFT43 surrounding basal bodies marked by Centrin-RFP (a, right) in a control cell. Similar pools are observed for GFP-IFT20 (b). GFP-IFT43 pools show reduced enrichment at basal bodies in Fuz KD cells (c). However, GFP-IFT20 is still appropriately localized under the same conditions (d). Note that Fuz KD cells exhibit a second phenotype of basal body clustering (yellow arrowheads in b and d; also see Gray et al. [2009]). Bars, 3 µm. (e) Quantitative comparison of GFP-IFT43 localization in control (Ctl) and Fuz KD cells. Each data point represents the mean of the mean intensities of all GFP-IFT43 pools in a cell normalized to the mean of the mean intensities of all Centrin foci. Fuz KD leads to a significant reduction in GFP-IFT43 localization to apical pools (Fuz KD [mean ± SD = 0.47 ± 0.20; n = 21 cells; six embryos] vs. control [mean ± SD = 0.67 ± 0.12; n = 21 cells; six embryos; P = 0.0003]). (f) A similar analysis of GFP-IFT20 shows no significant difference in localization between control and Fuz KD cells (Fuz KD [mean ± SD = 0.65 ± 0.20; n = 24 cells; six embryos] vs. control [mean = 0.66 ± 0.15; n = 22 cells; six embryos; P = 0.5308]). (g) A schematic model of IFT in control and Fuz KD axonemes.
Arts,
C14ORF179 encoding IFT43 is mutated in Sensenbrenner syndrome.
2011, Pubmed
Arts,
C14ORF179 encoding IFT43 is mutated in Sensenbrenner syndrome.
2011,
Pubmed
Banizs,
Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus.
2005,
Pubmed
Beales,
IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy.
2007,
Pubmed
Besschetnova,
Identification of signaling pathways regulating primary cilium length and flow-mediated adaptation.
2010,
Pubmed
Blacque,
Loss of C. elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised intraflagellar transport.
2004,
Pubmed
Bowes,
Xenbase: a Xenopus biology and genomics resource.
2008,
Pubmed
,
Xenbase
Cevik,
Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans.
2010,
Pubmed
Dai,
Fuz controls the morphogenesis and differentiation of hair follicles through the formation of primary cilia.
2011,
Pubmed
Davis,
TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum.
2011,
Pubmed
Deane,
Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles.
2001,
Pubmed
Dentler,
Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella.
2005,
Pubmed
Engel,
Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model.
2009,
Pubmed
Follit,
The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.
2006,
Pubmed
Gray,
The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis and mouse embryonic development.
2009,
Pubmed
,
Xenbase
Heydeck,
Planar cell polarity effector gene Fuzzy regulates cilia formation and Hedgehog signal transduction in mouse.
2009,
Pubmed
Huangfu,
Hedgehog signalling in the mouse requires intraflagellar transport proteins.
2003,
Pubmed
Iomini,
Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases.
2001,
Pubmed
Ishikawa,
Ciliogenesis: building the cell's antenna.
2011,
Pubmed
Jonassen,
Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease.
2008,
Pubmed
Kieserman,
High-magnification in vivo imaging of Xenopus embryos for cell and developmental biology.
2010,
Pubmed
,
Xenbase
Kim,
Planar cell polarity acts through septins to control collective cell movement and ciliogenesis.
2010,
Pubmed
,
Xenbase
Kozminski,
The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane.
1995,
Pubmed
Kozminski,
A motility in the eukaryotic flagellum unrelated to flagellar beating.
1993,
Pubmed
Li,
The small GTPases ARL-13 and ARL-3 coordinate intraflagellar transport and ciliogenesis.
2010,
Pubmed
Lyons,
The reproductive significance of human Fallopian tube cilia.
2006,
Pubmed
Marshall,
Flagellar length control system: testing a simple model based on intraflagellar transport and turnover.
2005,
Pubmed
Marshall,
Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control.
2001,
Pubmed
Mitchell,
A positive feedback mechanism governs the polarity and motion of motile cilia.
2007,
Pubmed
,
Xenbase
Omori,
Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8.
2008,
Pubmed
Ou,
Functional coordination of intraflagellar transport motors.
2005,
Pubmed
Pan,
Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors.
2006,
Pubmed
Park,
Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling.
2006,
Pubmed
,
Xenbase
Pazour,
Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella.
2000,
Pubmed
Pedersen,
Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling.
2008,
Pubmed
Pigino,
Electron-tomographic analysis of intraflagellar transport particle trains in situ.
2009,
Pubmed
Piperno,
Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects.
1998,
Pubmed
Qin,
Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.
2011,
Pubmed
Rix,
An Ift80 mouse model of short rib polydactyly syndromes shows defects in hedgehog signalling without loss or malformation of cilia.
2011,
Pubmed
Sawamoto,
New neurons follow the flow of cerebrospinal fluid in the adult brain.
2006,
Pubmed
Scholey,
Intraflagellar transport and cilium-based signaling.
2006,
Pubmed
Schrøder,
EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms.
2011,
Pubmed
Seo,
Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans.
2011,
Pubmed
Shindo,
Coordination of cell polarity during Xenopus gastrulation.
2008,
Pubmed
,
Xenbase
Singla,
Ofd1, a human disease gene, regulates the length and distal structure of centrioles.
2010,
Pubmed
Stubbs,
Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation.
2012,
Pubmed
,
Xenbase
Tran,
THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia.
2008,
Pubmed
Tsao,
Tetrahymena IFT122A is not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body.
2008,
Pubmed
Wanner,
Mucociliary clearance in the airways.
1996,
Pubmed
Woolner,
Imaging the cytoskeleton in live Xenopus laevis embryos.
2009,
Pubmed
,
Xenbase
