Fig 1: OFD1 is phosphorylated by PKA ASchematic representation of OFD1 domain organization, with the predicted PKA phosphorylation sites and the position of S735 and S899 in the human variant.BGST-fused, recombinant OFD1 polypeptides were used as substrates for in vitro phosphorylation assays using purified PKAc. GST-immobilized fusions were immunoblotted with an anti-phospho-(K/R)(K/R)X(S*/T*) antibody. A representative set of three independent experiments is shown. A Ponceau S staining of recombinant GST-OFD1 polypeptides is shown on the right panel. *Degradation products.CHEK293 cells transiently expressing flag-OFD1 or flag-S735A mutant were starved for 24 h and then treated with forskolin (FSK, 40 µM/1 h). Where indicated, cells were pre-treated with H89 (10 µM). Lysates were subjected to flag immunoprecipitation and immunoblot analysis with anti-phospho-PKA substrates and anti-flag antibodies. Anti-GFP IgG were used as control.DQuantitative analysis of the experiments shown in (C). A mean value ± SD of three independent experiments is shown. Student’s t test, *P < 0.05, **P < 0.01.ESame as in (C), with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2.FQuantitative analysis of the experiments shown in (E). A mean value ± SD of three independent experiments is shown. Student’s t test, *P < 0.05.GSchematic picture of the assembled TBC1D31 complex showing that following cAMP stimulation PKA phosphorylates the co-assembled OFD1. Source data are available online for this figure.
Fig 2: Lack of myosin VI alters the mobility and localisation of OFD1 at the centrioles ASuper-resolution analysis of OFD1 localisation at the centrioles. hTERT-RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti-OFD1 and anti-acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side.BTwo-colour SIM images in control and myosin VI-depleted cells illustrating the localisation and distribution of the distal appendage markers Cep164 and ODF2 at the centrioles, stained with anti-acetylated tubulin antibody. Representative images are shown; scale bar, 500 nm.CdSTORM super-resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI-depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm.D–FFRAP analysis of centriole-associated GFP-OFD1. hTERT-RPE cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 h, 6 µg/ml) and subjected to live-cell imaging. (D) Left: a scheme of the localisation of GFP-OFD1 and the centriole marker centrin1-dTomato. Right: a scheme of photobleaching and recovery of GFP-OFD1 at the centrioles. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n = 12 cells (Mock), n = 13 cells (MyoVI KD). (F) Quantification of the half-time of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ± SD. n = 41 cells (Mock), n = 31 cells (MyoVI KD), from three independent experiments. ns, not significant; ***P < 0.0005 by unpaired t-test. Source data are available online for this figure.
Fig 3: OFD1 is a component of the TBC1D31/praja2 complex AProtein–protein interaction network of physical interactors of OFD1, praja2 and TBC1D31. Node size represents the degree of association of the proteins (grey nodes) associated with the input proteins set (black nodes). Edge size is representative of the strength of the mapped interaction between the two connecting nodes.BThe bar plot reports the statistical significance of selected sets of Gene Ontology functional annotations enriched by the nodes in the network. Statistical significance is reported as ‐log10[P‐value] on the X axis, enriched functional annotations are reported on the Y axis.CLysates from HEK293 cells expressing GFP‐TBC1D31 and flag‐OFD1 were immunoprecipitated with anti‐flag or control IgG. Endogenous praja2 and the overexpressed transgenes were revealed by immunoblot analysis.DTotal lysates from mouse brain were immunoprecipitated with anti‐TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.ESchematic picture of the assembled TBC1D31/OFD1/praja2/PKA complex.FHEK293 cells were transiently co‐transfected with GFP‐TBC1D31 and flag‐OFD1. Cells were fixed and stained for flag and praja2. Source data are available online for this figure.
Fig 4: Myosin VI depletion leads to increased OFD1 and Cep164 recruitment at the centrioles ATotal lysates from hTERT-RPE1 cells were IP with anti-myosin VI antibody and an anti-rabbit IgG antibody (as control). IB was performed with anti-OFD1 and anti-myosin VI antibodies.BA scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the mother and daughter centrioles.C, DIF analysis of centriole-associated OFD1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI. Four days after transfection, cells were treated with nocodazole (1 h, 6 µg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. A scheme of the position of the markers used is depicted above. (C) Representative images, scale bar, 1 µm. (D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n = 148 cells; MyoVI KD, n = 199 cells, from four independent experiments. Daughter centrioles: Mock, n = 101 cells; MyoVI KD, n = 148 cells, from three independent experiments. ****P < 0.0001 by Mann–Whitney test.EIB analysis of hTERT-RPE cells treated with the indicated siRNAs with anti-myosin VI and anti-OFD1 antibodies. Anti-GAPDH was used as loading control.F, GIF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 h, 6 µg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 µm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mock, n = 147 cells; OFD1 KD, n = 146 cells; MyoVI KD, n = 150 cells; MyoVI + OFD1 KD, n = 151 cells, from four independent experiments. **P < 0.005; ****P < 0.0001 by Kruskal–Wallis test. Source data are available online for this figure.
Fig 5: Sequential recruitment of basal body anchoring proteins in Paramecium.(A) Paramecium transformants expressing either Cen2-GFP, CEP90-GFP, or OFD1-GFP (green) and stained for BB (poly-E antibodies, magenta) after 2 or 3 divisions in control cells (Ctrl) and upon OFD1, CEP90, or FOPNL depletion. Control-depleted paramecia displayed well-anchored BB organized in parallel longitudinal rows, with the GFP-emitted signal overlapping BB labeling. After OFD1, CEP90, and FOPNL depletion, BB pattern disorganization is observed. Scale bars = 20 µm and 2 µm (magnification). (B) Quantification of the GFP fluorescence of Cen2-GFP (orange), CEP90-GFP (purple), Cen2-GFP (orange), and OFD1-GFP (blue) on newly formed BB in control and upon Cen2, OFD1, FOPNL, CEP90, and Cen3 depletions. The dot plots show the average intensity of GFP fluorescence (n > 50 cells, 3 independent replicates). Error bars show SEM. Statistical significance was assessed by one-way ANOVA followed by Tukey’s post hoc test ****p < 0.0001. Source data can be found in S2 Data. (C) Schematic representation of the sequential recruitment of BB anchoring proteins leading to proper maturation of BB distal end. Cen2 arrives first at the BB followed by the interdependent recruitment of FOPNL, CEP90, and OFD1. This functional complex is required to allow Cen3 recruitment and BB docking. AU, arbitrary units; BB, basal body.
Supplier Page from MilliporeSigma for Anti-OFD1 antibody produced in rabbit