Abstract
The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to ‘segregate’ ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left–right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP–UBXN10 in ciliogenesis.
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Change history
01 February 2016
In the version of this Resource originally published, the sequence for the UBXN7 morpholino was reported incorrectly in the Methods section as 5′-TTTTGGATTCTCCACCCGAAGCCAT-3′. The correct sequence is 5′-ATGCGTCTCCGAGAGTCGCCATCTT-3′. This has been corrected in all online versions of the Resource.
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Acknowledgements
This work was supported by NIH grants R37-NS083524 and RO1-AG011085 to J.W.H. We would like to thank the Nikon Imaging Center (Harvard Medical School) for microscopy assistance. We would like to thank J. Wallingford (UT Austin) for the Xenopus laevis IFT-43 construct and M. Nachury and A. Nager (Stanford) for helpful discussions.
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Contributions
J.W.H. and M.R. conceived the project. M.R. prepared cell lines, performed immunofluorescence in cells and zebrafish, carried out cilia length measurements, performed and analysed AP–MS experiments (with the assistance of J.R.L.), and performed biochemical studies. M.S. performed cilia trafficking experiments under the direction of J.V.S. M.G. performed zebrafish experiments under the direction of W.G. E.L.H. performed the statistical analysis to determine P values for the MS data set. M.R. and J.W.H. wrote the paper with input from all authors.
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J.W.H. is a consultant for Biogen-Idec and Millennium: The Takeda Oncology Company.
Integrated supplementary information
Supplementary Figure 1 Proteomic analysis of the VCP-UBXD adaptor interaction network.
(a) Expression of C-HA/FLAG tagged baits in HEK-293T cells and comparison to endogenous proteins. Relative Expression levels were determined by quantification with a CCD camera in a FlourChem-M (Protein Simple). (b) Histograms representing bait abundance (based on average APSM) in α-FLAG and α-HA AP-MS experiments for all baits and the number of HCIPS identified for each bait. n = 4 LC-MS/MS experiments (derived form 2 technical replicates of two biological replicates). Error bars are mean +/− s.e.m from the four replicates. (c) Overlap of HCIPs in 6 AP-MS experiments. For this analysis, we used NWD ≥ 0.98, z ≥ 4, APSM ≥ 2. (d) Gene Ontology (GO) analysis for all unique proteins from Tiers 1-3. Analysis was performed with DAVID (http://david.abcc.ncifcrf.gov/). (e) Co-localization of C-HA/FLAG FAF2 with mitochondria. HeLa cells expressing C-HA/FLAG FAF2 were untreated or treated with 10 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 4 h. Cells were fixed and stained with α-HA and α-TOMM20. Scale bar is 10 μm.
Supplementary Figure 2 Localization of N or C-HA/FLAG tagged UBXD proteins, UFD1L, NPLOC4 and VCP in HeLa cells.
Localization of baits in HeLa cells was visualized by α-HA staining. Scale bar (shown on VCP N-HA/FLAG merged image) is 10 μm.
Supplementary Figure 3 Topology of UBXD complexes.
(a-g) Consolidated interaction maps of HCIPs identified across N and C-tag α-FLAG and α-HA APMS studies. (a) UFD1L, (b) UBXN4, (c) UBXN2A, (d) FAF1, (e) ASPSCR1, (f) NSFL1C and (g) UBXN11, (h) UBXN6, (i) NPLOC4. Legend describes criteria used for determining HCIPs.
Supplementary Figure 4 UBXN10 interacts with the Intraflagellar Transport B (IFT-B) complex that controls anterograde trafficking into cilia.
(a) HA-UBXN10 and MYC-IFT-B were in vitro translated in HeLa lysates. α-MYC immunoprecipitates were resolved by SDS-PAGE and probed for associated HA-UBXN10. GFP was used as a negative control. UNC13B, which is not an IFT-B subunit, was also validated in this manner. (b) A subset of IFT-B proteins are present in the HeLa IVT lysate as determined by immunoblotting. (c) MYC-IFT25 was in vitro translated in HeLa IVT lysate and immunoprecipitated with MYC agarose. Bound complexes were analyzed by LC-MS/MS. IFT-B proteins in HeLa lysate that interacted with MYC-IFT25 are shown. (d) MYC-GFP, IFT25 and IFT27 were in vitro translated in HeLa IVT lysate and immunoprecipitated with MYC agarose. Immunoprecipitates were probed for IFT27. MYC-IFT27 binds IFT25 in HeLa lysate. (e) UBXN10 binds specifically to the CLUAP1 subunit of IFT-B. 35S-labelled-IFT-B subunits were translated in rabbit reticulocyte lysates and incubated with GST alone or GST-UBXN10 or GST-VCP purified from E.Coli. GST pull-downs were resolved by SDS-PAGE, stained with Coomassie and subjected to autoradiography. ∗: 35S-labelled- IFT52 was inadvertently loaded twice in the input gel alone. The longer exposure of the autoradiograph shows weaker (non-specific) binding to IFT46, 52 and 57 by GST, GST-UBXN10 and GST-VCP.
Supplementary Figure 5 VCP-UBXN10 localizes to cilia.
(a) GFP-UBXN10 localizes to cilia in LLC-PK1. Scale bar is 10 μm. (b) Localization of VCP, NPLOC4, UBXN1 and UBXN7 in LLC-PK1 cells. LLC-PK1 cells were infected with virus encoding the indicated GFP tagged proteins. Stable cell lines were serum-starved for 48 h, fixed and stained with acetylated tubulin. Scale bar is 10 μM. (c) The UBX domain of UBXN10 was modeled onto the FAF1-VCP co-crystal structure (PDB ID: 3QWZ) using PHYRE. The location of the TMEVPR loop in UBXN10 is represented.
Supplementary Figure 6 Depletion of UBXN10 inhibits cilia formation.
(a) LLC-PK1 GFP-UBXN10 cells were transfected with siRNAs targeting VCP or UBXN10. Cells were induced to ciliate by serum deprivation, fixed and stained with acetylated tubulin. The number of ciliated cells and the number of GFP-UBXN10 positive cilia were quantified. n = 131 (Control), 124 (siUBXN10-3), 156 (siVCP-7) cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance was calculated using unpaired, two-tailed Students t-test. P values compared to Control are shown. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (b) UBXN10 was stably depleted in hTERT-RPE1 using two separate pLKO-based short hairpin RNAs. Cells were selected with Puromycin, induced to ciliate and the number of ciliated cells was quantified based on acetylated tubulin staining. n = 98 (shGFPl), 115 (sh114), 103 (sh909) cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance was calculated using unpaired, two-tailed Students t-test. P values compared to shGFP are shown. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (c) UBXN10 knockout hTERT-RPE1 cells generated via CRISPR-CAS9 have impaired ciliogenesis. 40X images showing a larger field of view. Expression of GFP-UBXN10 in the knockout cell line reinstates ciliogenesis. Cells were stained with pericentrin and acetylated. Scale bar is 10 μm. (d) Cells were treated with either 50 μM NMS-873 for 5 min or 5 μM NMS-873 for 4 h, extracts were generated and subjected to SDS-PAGE and immunoblotting with α-ubiquitin antibodies.
Supplementary Figure 7 Inhibition of VCP leads to destabilization of IFT-B and altered rates of trafficking within the primary cilium.
(a) Acute treatment with 50 μM NMS-862 or NMS-873 leads to an increase in both anterograde and retrograde IFT-88 EYFP velocities. Treatment with 5 μM NMS-862 or NMS-873 for short (<5 min) time periods does not produce the same phenotype. The fluorescence image time series was subjected to kymograph analysis in ImageJ. n (anterograde, retrograde) from left to right = 54, 56, 61, 63, 50, 62, 53, 60, 68, 56 cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance calculated using ANOVA, P values compared to DMSO for anterograde or retrograde transport are shown. N.S., not significant. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (b) Treatment of LLC-PK1 cells with NMS-873 leads to a shortening of cilia over time. Cells were treated with 5 μM NMS-873 for indicated times and stained for acetylated tubulin. The length of the cilia were measured based on acetylated tubulin staining on MetaMorph. (c) Kymographs derived from TIRF microscopy of IMCD EYFP-IFT88/UBXN10-mCHERRY treated with DMSO or 50 μM NMS-873 for <5 min. Only cilia that displayed motile EYFP-IFT88 particles were measured for this study. The fraction of these cilia that also exhibited observable UBXN10-Cherry motility was then determined. Treatment with NMS-873 resulted in a loss of UBXN10 trafficking within cilia. n = 75 (DMSO) and 82 (NMS-873). Scale bars in time (2 s) and distance (2 μm) are shown. (d) Inhibition of VCP leads to destabilization of the IFT-B complex. Ciliated hTERT RPE1 cells were treated with 5 μM of NMS-873 for 4 h. Cell lysates were fractionated by gel filtration, resolved by SDS PAGE and probed for the indicated proteins. Treatment with NMS-873 causes CLUAP1 destabilization from higher molecular weight fractions. (e) hTERT-RPE1 cells were transfected with siRNAs to IFT25 or IFT27. Lysates were resolved on SDS PAGE and immunoblotted to show antibody specificity and observed molecular weights of the IFT-25 and IFT27. (f) LLC-PK1 cells were treated with 5 μM of NMS-873 in a time course experiment. Cell lysates were resolved on SDS PAGE and immunoblotted with the indicated antibodies. UBXD adaptor levels begin to decline at 4 h post treatment. (g) GFP-UBXN10 LLC-PK1 cells were treated with 5 μM of NMS-873 in a time course experiment. Cell were fixed and stained for GFP and acetylated tubulin and the intensity of GFP-UBXN10 within cilia was determined using Metamorph. Treatment with NMS-873 causes a decrease in GFP-UBXN10 in cilia within an hour. n = 56 (DMSO), 72 (1 h), 45 (2 h), 31 (4 h) cells pooled from 2 independent experiments. Maxima, centre, minima and quartiles (Q1,2,3) for each sample from left to right: (68, 33, 8, 14, 20, 29), (38, 16, 6, 9, 12, 19) (29, 13, 3, 7, 12, 17), (21, 9, 3, 8, 9, 18). Error bars represent mean ± s.d. Statistical significance calculated using ANOVA, P values compared to DMSO are shown. ∗∗P ≤ 0.01.
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UBXN10 exhibits IFT-like transport within cilia.
Live cell imaging of GFP-UBXN10 demonstrating IFT-like particle motility within cilia in hTERT-RPE1 cells. (MOV 1666 kb)
UBXN10 and IFT-B migrate as part of the same complex within cilia.
Dual colour TIRF microscopy of IMCD cells expressing EYFP-IFT88 and UBXN10-mCHERRY to visualize co-migration within cilia. (MOV 1383 kb)
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Raman, M., Sergeev, M., Garnaas, M. et al. Systematic proteomics of the VCP–UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis. Nat Cell Biol 17, 1356–1369 (2015). https://doi.org/10.1038/ncb3238
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DOI: https://doi.org/10.1038/ncb3238
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