XB-ART-46091J Cell Biol. July 9, 2012; 198 (1): 37-45.
Control of vertebrate intraflagellar transport by the planar cell polarity effector Fuz.
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.
PubMed ID: 22778277
PMC ID: PMC3392940
Article link: J Cell Biol.
Grant support: R01 GM074104 NIGMS NIH HHS , Howard Hughes Medical Institute
Genes referenced: fuz ift20 ift43 map7 plin1 tbx2
Disease Ontology references: ciliopathy
External Resources: GO Terms referenced: intraciliary anterograde transport
Article Images: [+] show captions
|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. (a–d) 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. ). 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.|