Fig 2: Inhibition of VCP leads to destabilization of IFT-B and altered rates of trafficking within the primary cilium(a) Inhibition of VCP causes cilia shortening. Cells were treated with 5 μM (1X) or 50 μM (10X) DBeQ. n = 66 cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance calculated using ANOVA, p values shown. n.s, not significant **: p≤ 0.01.(b) Acute treatment with DBeQ increases anterograde and retrograde IFT-88 velocities. n = 56 cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance calculated using ANOVA, p values shown. n.s, not significant. **: p≤ 0.01.(c) VCP inhibition uncouples UBXN10 from IFT-B. Cells were treated with DMSO or 50 μM NMS-873 for < 5 min. Cilia that displayed motile EYFP-IFT88 particles were measured. The fraction of such cilia with observable UBXD3-Cherry motility was determined. n = 75 (DMSO) and 82 (NMS-873) cells.(d) Acute treatment with NMS-873 decreases anterograde and retrograde IFT-43 velocities. n = 40 cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance calculated using ANOVA, p values shown. **/*: p≤ 0.01, 0.05.(e,f) VCP inhibition causes loss of ciliary BBS5. Cells were treated with 5 μM NMS-873 and the intensity of BBS5 within cilia was quantified. n (left to right) = 75, 68, 69, 33, 42 cells pooled from 2 independent experiments. Maxima, centre, minima and quartiles (Q1,2,3) left to right : (100, 60, 22, 43, 70, 73), (77, 53, 20 37, 51, 71), (49, 37, 18, 37, 39, 41), (47, 31, 20, 27, 29, 36), (39, 31, 8, 28, 34, 37). Error bars represent mean ± s.d. Statistical significance calculated using ANOVA, p values shown. **/*: p≤ 0.01, 0.05. Scale bar, 5 μm.(g) VCP inhibition leads to destabilization of IFT-B complex. Cells were treated with 5 μM NMS-873 for 4 h. Lysates were fractionated by gel filtration and probed for the indicated proteins. IFT25-27 specific bands determined by siRNA knockdown (Supplementary Fig. 7c).(h,i) Depletion of zebrafish Ubxn10 causes defects in left/right asymmetry. Whole-mount in situ staining with the pan-cardiomyocyte marker cmlc2 demonstrating position of heart tube. Scale bar, 200 μm (h). Summary of 3 independent MO experiments. p values calculated using Fishers exact test (i).(j) Model. Uncropped images of blots are shown is Supplementary Fig. 8.

Fig 3: Proteomic Analysis of the VCP-UBXD adaptor interaction network(a) Domain organization of VCP and UBXD proteins used as baits in the AP-MS studies. (D1, D2: ATPase domains, PUB: Peptide N-glycosidase/Ubiquitin associated, ThF: Thioredoxin like Fold, UBA: Ubiquitin associated, UIM: ubiquitin interaction motif, UBL: Ubiquitin –Like, SEP: Shp, EYC, p47, NZF: Npl4 Zinc Finger, UT3: Ufd1 Truncation 3).(b) Pipeline for interaction proteomics and localization experiments.(c) Localization of selected baits in HeLa cells visualized by α-HA staining. Scale bar, 10 μm.(d) Organization of interactions observed in 6 AP-MS experiments into categories (Tiers) based on the criteria indicated under “categories”.(e) Heat map of HCIPs (represented by APSM) identified for all baits. HCIPs were filtered using a NWD ≥ 0.98, Z ≥ 4, APSM ≥ 2 between biological duplicates. Hierarchical clustering was carried out based on interaction profiles. Selected complexes or clusters have been highlighted in detail and relevant members are color-coded.

Fig 4: Topology of UBXD and VCP complexes(a-g) Consolidated interaction maps of HCIPs identified across N and C-tag α-FLAG and α-HA APMS studies. (a) UBXN10, (b) UBXN7, (c) FAF2, (d) UBXN1, (e) UBXN2B, (f) UBXN8 and (g) VCP. Legend describes criteria used for inclusion.

Fig 5: Overview of the VCP-UBXD adaptor network(a) Comparison of interacting proteins for 12 UBXD, UFD1L, and NPLOC4 proteins identified here with those reported in BioGRID. (AP-MS and IP-IB data in BioGRID available for 13 adaptors, yeast two-hybrid data available for 6 adaptors). See Supplementary Table 3.(b) Comparison of interacting proteins for UBA-UBXD proteins found previously (Reference 6).(c) Interactions observed among UBXD, UFD1L, and NPLOC4 proteins in the data reported here (left panel) and reported previously in BioGRID (right panel).(d) Integrated interaction map of the VCP-UBXD network. HCIPs were filtered using NWD ≥ 0.98, Z ≥ 4, APSM ≥ 2 between α-FLAG biological duplicates and additionally verified in α-HA AP-MS. For clarity gene names have been omitted, except when HCIPs are shared between UBXD proteins. Interactions between UBXD adaptors have been omitted for clarity.