Fig 2: FAM83D regulation during the cell cycle ANocodazole‐synchronised mitotic FAM83D GFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ‐phosphatase. Asynchronous (AS) cells were used as a control. Whole‐cell extracts (input) and IP samples were subjected to SDS–PAGE and subsequent immunoblotting (IB) with the indicated antibodies.B–DSTLC‐synchronised mitotic wild‐type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B), medium ± MG132 (C) or medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies.EWild‐type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake‐off (M). AS and M cells were incubated in media containing combinations of RO‐3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO‐3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS–PAGE, before IB with the indicated antibodies.FSTLC‐synchronised mitotic wild‐type U2OS cells were subjected to qRT–PCR analysis using primers for FAM83D, HMMR, CSNK1A1 and CCNB1. AS cells were used as a control. Error bars, SEM; *P < 0.01; Student's t‐test. n = 3.GSchematic representing the predicted effects of HMMR knockout on FAM83D–CK1α delivery to the mitotic spindle. In the absence of HMMR, no FAM83D and, by extension, no CK1α can localise to the spindle.HSTLC‐synchronised mitotic wild‐type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control.I, JThe cells described in (H) were STLC‐synchronised in mitosis and subjected to immunofluorescence microscopy with an anti‐HMMR antibody (I) or an anti‐CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 μm.Data information: All blots are representative of at least three independent experiments. Source data are available online for this figure.

Fig 3: Schematic of the CRISPR/Cas9 gene editing strategies, and retrovirally expressed nanobody‐based systems used in this studySchematic detailing the CRISPR/Cas9 gene editing strategies employed to generate the indicated cell lines (left‐hand side). The schematic on the right‐hand side details the retrovirally expressed nanobody‐based degradation (VHL‐aGFP.16), and targeting (aGFP.16‐CK1) strategies used in this study.

Fig 4: FAM83H interacts with NCK adaptor proteins.A Schematic diagram showing FAM83 proteins interact with CK1 isoforms and have unique interactors.B HEK-293 Flp-In T-REx cells that express GFP-FAM83 proteins under a doxycycline inducible promoter were treated with 20 ng/mL doxycycline for 24 h and GFP was pulled down from lysates then cell extracts (input) and pull downs (IP) were analysed by immunoblotting (IB). n = 3.C U2OS FAM83H-/- cells were transiently transfected with vectors encoding GFP, GFP-FAM83H and/or mCherry-UNC45A and lysates subjected to anti-GFP pull downs. Cell extracts (input) and pull downs (IP) were then analysed by Western blotting. n = 2.D Empty FLAG vector (vector), FLAG-FAM83HS287X, FLAG-FAM83HQ452X, FLAG-FAM83HE694X and FLAG-FAM83HWT were transiently transfected into U2OS FAM83H-/- cells. Untransfected U2OS FAM83H-/- cells (−) were used as an additional control. Anti-FLAG immunoprecipitations were performed on lysates and cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 3.E FLAG empty vector (vector), FLAG-FAM83HS342T, FLAG-FAM83HG557C, FLAG-FAM83HE694X and FLAG-FAM83HWT were transiently transfected into HEK-293 wild type cells. Anti-FLAG pull downs were performed on lysates then cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 1.

Fig 5: Wild type FAM83H and disease mutants co-localise with CK1, but mutants do not colocalise with NCK1.A U2OS FAM83H-/- cells transiently expressing GFP, GFP-FAM83H, mCherry-NCK1 or mCherry-NCK2, as indicated, were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged using the DeltaVision widefield fluorescence microscope then images were deconvolved and processed using OMERO (12). Scale bar represents 10 μm. n = 3B As A but U2OS FAM83H-/- cells were transiently transfected with FLAG-FAM83HWT (WT), FLAG-FAM83HS287X (S287X), FLAG-FAM83HQ452X (Q452X), FLAG-FAM83HE694X (E694X), or no FLAG construct (−) and co-transfected with mCherry-CK1α. Scale bar represents 10 μm. n = 3C As B but with mCherry-NCK1 instead of mCherry-CK1α. n = 2D U2OS FAM83H-/- cells transiently expressing FLAG (vector), FLAG-FAM83HS342T, FLAG-FAM83HG557C, FLAG-FAM83HWT and mCherry-CK1α were fixed and stained with anti-FLAG antibodies and DNA stained with DAPI. Cells were imaged using DeltaVision widefield fluorescence microscope. Scale bar represents 10 μm. n = 2E As D but mCherry-NCK1 was used instead of mCherry-CK1α. Scale bar represents 10 μm. n = 1