Abstract
Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11–Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
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Change history
12 March 2015
In the version of this Article originally published Fig. 3g was incorrectly labelled. The top right panel should have been labelled 'EHD1' in red and the entire of 3g should have been labelled 'Rab8a+8b siRNA' in black. The image is corrected in all online versions of the Article.
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Acknowledgements
We thank K. Nagashima and A. Kamata for help with IEM and EM, C. Lamont for data analysis, S. Lockett for help with SIM imaging, S. Specht for help with cell culture, A. Peden for SNAP29 antibodies, J. Goldenring for Rab11 antibodies, Z. Sun for the transgenic line and S. Burgess for assistance raising zebrafish embryos. We are grateful to J. Donaldson for critical reading of this manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. This project has also been funded in part with federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.
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Q.L. and C.I. carried out most experiments with help from C.J.W. (RNAi, fluorescence imaging and PCR with reverse transcription), C.O. (SIM imaging), P.A.P., S.L., I.O.D. and Y-S.H. (in situ expression in zebrafish), U.B. (EM and CLEM), V.W. (immunoblotting), J.S. (zebrafish MO experiments) and A.C. (cell line generation and RNAi rescue experiments). S.C. and J.R. provided reagents. S.C., C.O., J.L-S., and P.K.J. discussed the results and commented on the manuscript; C.J.W. and C.I. wrote the paper with suggestions from Q.L. and C.O. C.J.W., Q.L. and C.I. conceived and designed the research.
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Integrated supplementary information
Supplementary Figure 1 EHD1 and EHD3 functions in ciliogenesis and localizes in the ciliary pocket.
a. EHD transcripts were reduced by > 90% as determined by qRT-PCR using two different siRNA oligonucleotides (#1 and #2) for each EHD family member. Averages of three technical replicates from n = 1 experiment are shown. b,c. Quantification of ciliation in RPE cells treated with siRNA as described in Fig 1b. b. Mean ± SEM from n = 3 independent experiments (> 300 cells per treatment) is shown. Two tailed t-test analysis compared with siControl. c. Pooled averages across a single experiment. d. Representative images of RPE cells quantified in Fig 1c. Cells were treated for 6h with siEHD1#1, transiently transfected with siRNA resistant (Res) GFP-EHD proteins or GFP and after 48h cells were serum starved for 24h. Cells were stained with Actub and pericentrin (PCNT) antibodies and imaged by epifluorescence microscopy. e. Quantification of IFT20 fluorescence intensity at the M-centriole in cells treated as described in (d). IFT20 localization was determined as described in Fig 5g. Mean ± SEM from n = 3 independent experiments is shown (total cells counted across all experiments: GFP, 34 cells; GFP-EHD1, 35 cells, GFP-EHD3, 52 cells). Two tailed t-test analysis compared with GFP. f. Quantification of RPGRIP1L fluorescence intensity at the transition zone in cells treated as described in (d). RPGRIP1L transition zone (TZ) localization was determined as described in Fig. 5b. Mean ± SEM from n = 3 independent experiments is shown (total cells counted across all experiments: GFP, 28 cells; GFP-EHD1, 36 cells, GFP-EHD3, 37 cells). Two-tailed t-test analysis compared with GFP. g. Representative image of RPE cells transiently expressing GFP-EHD3 and Smo-tRFP, serum starved for 24h, stained with anti-EHD1 and imaged by epifluorescence microscopy. Scale bar: 2.5 μm. h. Representative image of RPE cells stably expressing GFP-Rab8a, serum starved for 24h and stained with anti-EHD1 and anti-Actub antibodies. Scale bar: 5 μm. i. Representative images of IMCD3 cells transiently expressing GFP-EHD1 (top panel) or GFP-EHD3 (bottom panels), serum starved for 24h and stained with anti-Actub antibody and DAPI and imaged by epifluorescence microscopy. Scale bar: 5 μm. j. Representative images from correlative light and electron microscope (CLEM) imaging of RPE GFP-EHD1 cells transiently expressing tRFP-Rab8a. Following fixation cells were imaged by spinning disk confocal imaging and processed for electron microscopy. Region of interest with cells from fluorescence imaging were identified in electron microscopy sections. The same cilium detected by electron microscopy and fluorescence microscopy (dotted box) is shown on the right panels. n: nuclei. Scale bars: 2 μm. k,l. Representative SIM images of a RPE GFP-EHD1 cells transiently expressing tRFP-Rab8a (k) and tdTomato-Inversin (tdTom-Inversin) (l) for 72h with serum starvation for 24h. Scale bar: 500 nm. ∗ P < 0.05,∗∗∗ P < 0.0001. Statistics source data for Supplemental Fig 1b,e,f can be found in Supplementary Table 2.
Supplementary Figure 2 Expression of Ehd1 and Ehd3 in zebrafish.
a. Western blot analysis of lysates from uninjected embryos or embryos injected with ehd1a + b (left panel) or ehd3 (right panel) MO with and without human EHD1 or EHD3 mRNA. Knockdown efficiency and rescue protein expression levels from western blots band densities were quantified and shown in bar graphs. Note that in these representative rescue experiments, embryos co-injected with ehd1MO and hEHD3 mRNA had < 20% Ehd1 protein left and overexpressed EHD3 at > 15 fold. Similarly, embryos co-injected with ehd3MO and hEHD1 mRNA had < 10% Ehd3 protein left and overexpressed EHD1 at > 8 fold. Un-cropped images are shown in Supplemental Fig 6. b. Quantification of kinocilia number in neuromasts of ehd1 and ehd3 morphants. Note that ehd3 morphants present a partial neuromast defect with an average of 2.5 ± 1.8% kinocilia per neuromast versus 0.9 ± 1.3% in ehd1 morphants. Averages ± SD are pooled data from 3 independent experiments from control n = 19 neuromasts, ehd1MO n = 15 neuromasts, and ehd3 MO n = 16 neuromasts. Two tailed t-test analysis, ∗∗∗ P < 0.0001. Statistics source data for Supplemental Fig. 2b can be found in Supplementary Table 2. c. Standard whole mount in situ hybridizations performed at 48hpf and 72hpf with ehd1 and ehd3 probes designed for the 3’UTR regions. The ehd1 probe was predicted to recognize both ehd1a and ehd1b transcripts. ehd1 mRNA expression was detected prominently in the eye and otic vesicles (OV) at 48hr and 72hpf whereas ehd3 mRNA was mainly detected in the eye at 48 and 72hpf. Insets show 48 hpf expression in OV region. d. Sections of retinae from 4dpf control or MO injected embryos stained with anti-Ehd1 or anti-Ehd3 antibodies and DAPI. Uninjected and MO injected retinae sections were stained at the same time demonstrating specific expression of Ehd1 and Ehd3 in the retinae. Scale bar: 10 μm. e. Representatives images of otic vesicles in MO injected embryos at 3dpf stained with anti-Actub and anti-Ehd1 or anti-Ehd3 antibodies as described in (d). Scale bar: 10 μm.
Supplementary Figure 3 GFP-EHD1 and tRFP-Rab8 localizations in RPE cells following serum starvation.
Full cell images of RPE GFP-EHD1 cells transiently expressing tRFP-Rab8a after serum starvation shown in Fig 3c. Cropped regions were shown in square boxes and the nuclear region is marked by blue dotted lines. Scale bar: 10 μm.
Supplementary Figure 4 Representative electron micrographs of M-centrioles from RPE cells and zebrafish photoreceptors.
a. Additional representative TEM images of RPE cells treated with siControl in presence of serum, or siEHD1 (upper panel) or siRab8 (lower panel) after 24h starvation as shown in Fig 4a and quantified in Fig 4b. Scale bar: 250 nm. b. Additional representative TEM images of zebrafish photoreceptor cells treated with ehd1 + 3 MO and Rab8 MO as described in Fig 4g and quantified in Fig 4h. Scale bar: 250 nm.
Supplementary Figure 5 B9D2-GFP is recruited to the M-centriole after the ciliary membrane receptor 5-HT6-tRFP in RPE cells following serum starvation.
RPE B9D2-GFP cells transiently expressing 5HT6-tRFP, serum starved for 1h and imaged live by spinning disk confocal microscopy. Images show single xy-planes from z-stacks. Scale bar: 2 μm.
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GFP-EHD1 localizes to developing ciliary membranes prior to tRFP-Rab8a.
Time-lapse video of a RPE cell expressing GFP-EHD1 and tRFP-Rab8a. Spinning disk confocal images were taken every 5 min after 1hr starvation. (MOV 1351 kb)
GFP-EHD1 localizes to the developing ciliary membrane with Smo-tRFP.
Time-lapse video of a RPE cell expressing GFP-EHD1 and Smo-tRFP. Spinning disk confocal images were taken every 10 min after 1hr starvation. (MOV 440 kb)
GFP-Rab8 is recruited to the developing ciliary membrane after Smo-tRFP.
Time-lapse video of a RPE cell expressing GFP-Rab8 and Smo-tRFP. Spinning disk confocal images were taken every 10 min after 1hr starvation. (MOV 125 kb)
IFT20-GFP is recruited after pre-ciliary membrane accumulation of Smo-tRFP.
Time-lapse series showing a RPE cell expressing IFT20-GFP and Smo-tRFP. Spinning disk confocal images were captured every 10 min after 1hr starvation. (MOV 262 kb)
IFT20-tRFP localizes to developing cilia prior to GFP-Rab8a recruitment.
Time-lapse video of a RPE cell expressing GFP-Rab8 and IFT20-tRFP. Spinning disk confocal images were captured every 20 min after 1hr starvation. (MOV 310 kb)
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Lu, Q., Insinna, C., Ott, C. et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol 17, 228–240 (2015). https://doi.org/10.1038/ncb3109
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DOI: https://doi.org/10.1038/ncb3109
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