Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. SSTR3 fused to the fluorescent protein mNeonGreen (NG) at its intracellular C-terminus and a biotinylation Acceptor Peptide (AP) tag at its extracellular N-terminus was expressed under the control of an attenuated EF1α promoter (pEF1α Δ ) in wild type (WT) and Arl6 -/- IMCD3 cells. Cells were treated with or without somatostatin-14 (sst) for 2 h, then fixed and stained for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). AP SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 2μm (inset). In WT cells, the ciliary SSTR3 signal decreases over the experimental time course. In Arl6 -/- cells, SSTR3 fails to exit cilia and an increase in the ciliary Ub level is detected. As a control, Arl6 -/- cells that did not express SSTR3-NG were tested; no increase in ciliary Ub levels was observed upon addition of sst. B. The fluorescence intensity of the Ub channel in the cilium was measured in each condition and the data are represented as violin plots. The thick bar indicates the median and the dotted lines the first and third quartiles. An 11-fold increase in ciliary Ub signal is observed upon addition of sst to SSTR3-expressing cells. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. WT or Arl6 -/- IMCD3 cells stably expressing AP SSTR3 NG and the biotin ligase BirA targeted to the ER lumen (BirA-ER) were transiently transfected with HA-tagged ubiquitin (HA-Ub). 10 µM biotin was added to cells 24 h post transfection for maximal biotinylation of AP SSTR3 NG and, after another 18 h, cells were treated with sst (10μM) for indicated times. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated three times and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. E. IMCD3 cells of the indicated genotypes were treated with the Smoothened agonist SAG or the vehicle DMSO for 2h. Cells were then fixed and stained for acTub and Ub. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). Activation of Hh signaling promotes a detectable increase in ciliary Ub levels only in Arl6 -/- cells. F. Violin plots of the fluorescence intensity of the Ub channel in the cilium in each condition are shown. Asterisks indicate ANOVA significance value. ****, p= <0.0001.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Control, Staining, Ubiquitin Proteomics, Fluorescence, Expressing, Stable Transfection, Transfection, Western Blot, Quantitation Assay, Activation Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3 cells of the indicated genotypes expressing AP SSTR3 NG were treated with somatostatin-14 for 2 h. Cells were fixed and stained for AcTub (magenta) and with antibodies specific for the lysine 63 (UbK63) or lysine 48 (UbK48) Ub chain linkages (yellow). SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). B. The fluorescence intensity of the UbK48 and UbK63 channels in the cilium are represented as violin plots. A 14-fold increase in ciliary Ub abundance is detected with the K63Ub linkage-specific antibody. Asterisks indicate ANOVA significance value. ****, p= <0.0001. No ciliary signal is detected with the K48Ub linkage-specific antibody. C. Arl6 -/- IMCD3-[ AP SSTR3; BirA-ER] cells were transfected with the HA-tagged ubiquitin variants WT, noK0 (all seven acceptor lysine residues mutated to arginine) or K63 (where all lysine residues are mutated to arginine except for K63). Biotin was included in the culture medium and cells were treated with sst for 0 or 10 min. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated four times and for each Ub variant, Ub-SSTR3 signals were normalized to the value at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. Asterisks indicate ANOVA significance value. *** p = < 0.001; ** p=<0.01.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Expressing, Staining, Fluorescence, Transfection, Ubiquitin Proteomics, Western Blot, Control, Quantitation Assay, Variant Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3-[pEF1α Δ - AP SSTR3 NG ] cells of the indicated genotypes were treated with sst for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). SSTR3 NG was visualized through the intrinsic fluorescence of NG (cyan). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. SSTR3 exit is blocked in Arl6 -/- cells regardless of the β-Arrestin2 genotype but the ciliary Ub signal is only evident when β-Arrestin2 function is intact. Scale bar: 5μm (main panel), 1μm (inset). B. Violin plots representing the ciliary levels of Ub under the indicated conditions. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. IMCD3 WT, knockout for Arl6 only and double knockout for Arl6 and the β-Arrestin2 gene Arrb2 were treated with SAG for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 1μm (inset). D. Violin plots representing the ciliary Ub levels. Asterisks indicate ANOVA significance value. ****, p= <0.0001; **, p= <0.01. The appearance of a Ub signal in cilia in Arl6 -/- cells depends on β-Arrestin2. E. IMCD3-[ AP SSTR3; BirA-ER] cells of the indicated genotypes stably expressing AP SSTR3 NG and BirA-ER were transfected with HA-Ub, biotin was added to the medium and cells were treated with sst for indicated times. Biotinylated SSTR3 was captured from cell lysates under denaturing conditions on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6, β-Arrestin2 to verify genotypes and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. F. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in E was repeated twice and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Staining, Ubiquitin Proteomics, Fluorescence, Knock-Out, Double Knockout, Stable Transfection, Expressing, Transfection, Western Blot, Control, Quantitation Assay