Generation of constructs<\/p>\n
Constructs were developed using a modified overlap cloning technique60<\/a>. Flexible linkers, consisting of glycine\u2013serine repeats (GGGGS), with lengths ranging from 18 to 33 amino acids, were inserted between the coding sequences for fluorescent proteins or luciferases and those of receptors, transducers, biosensors or adaptor proteins. The creation of SmBiT-\u03b2-arrestin IP6, AP2 and clathrin mutants used a previously published overlap cloning strategy with SmBiT. The 2xFYVE-mKO construct was created by integrating Cyto-mKO into the 2xFYVE-LgBiT vector via overlap cloning.<\/p>\n Generation of \u03b2-arrestin-split\u00a0GFP<\/p>\n \u03b2-Arrestin-split\u00a0GFP constructs were derived from pcDNA3.1-GFP(1\u201310) (Addgene no. 70219) and pEGFP-GFP11-Actin (Addgene no. 181966) and cloned into either SmBiT-\u03b2-arrestin or \u03b2-arrestin-SmBiT.<\/p>\n Generation of stable cell lines<\/p>\n \u03b2-Arrestin knock-in\u00a0cell\u00a0lines were made by Cyagen. In short, the Arrb1\u20132 (C terminal\u00a0linker-GFP11) knock-in HEK293T cells wereseeded into six-well plates and cultured for 24\u2009h. Cas9 nuclease (0.2\u2009nmol) and guide RNAs (0.1\u2009nmol; sequences in Supplementary Table 1<\/a>) were pre-incubated to assemble ribonucleoprotein complexes, and 0.5\u2009nmol of donor vector was electroporated into the target cells. Within 24\u201348\u2009h post-electroporation, the cell pool was sorted and tested for the cell condition using DAPI-negative staining. Cell pools with high editing efficiency were selected for monoclonal preparation. Single cells were deposited into 96-well plates by limiting dilution. After approximately 2 weeks of culture, well-growing single clones in good condition were expanded, and genomic DNA was analysed by PCR (primer listed in Supplementary Table 1<\/a>) and sequencing (Genscript) to confirm precise C\u00a0terminal-linker-GFP11 integration.<\/p>\n Mutagenesis of \u03b2-arrestin IP6 and IDR mutants<\/p>\n Mutation generation was performed using the QuikChange site-directed mutagenesis kit (Agilent). The mutations produced included \u0394IP6-N, \u0394IP6-C, \u0394IP6-N\u2013C, \u0394IP6-NT, \u0394IP6-NT\u2013C, \u0394IDR and \u0394CT.<\/p>\n Opto-\u03b2-arrestin constructs<\/p>\n Opto-\u03b2-arrestin constructs were designed on the basis of those from a past work35<\/a>. pHR-mCh-Cry2WT (Addgene no. 101221) and pHR-FUSN-mch-Cry2WT (Addgene no. 101223) were cloned into the pcDNA backbone. Overlap cloning was then used to generate all opto-\u03b2-arrestin constructs.<\/p>\n Immunofluorescence<\/p>\n HEK293T cells were fixed with 4% PFA for 20\u2009min and then washed three times with phosphate-buffered saline (PBS). Cells were incubated with donkey serum blocking buffer with 0.1% Triton X-100 for 1\u2009h and then washed three times with PBS. Cells were then stained with primary antibodies with \u03b2-arrestin 1 (BD Biosciences, 610550, 1:100) or \u03b2-arrestin 2 (Abnova, M06J, 1:500) overnight for 4\u2009\u00b0C, and then washed three times with 1\u00d7\u2009PBS. Secondary antibodies (1:500) and Hoechst 33342 (1:1000) were then applied for 2\u2009h at room temperature and washed three times with 1\u00d7\u2009PBS.<\/p>\n BRET and split nanoluciferase assays<\/p>\n For BRET and split luciferase assays, HEK293T cells were cultured in six-well plates and transiently transfected using polyethylenimine (PEI) as outlined previously. The \u03b2-arrestin association was analysed using split NanoLuc components (that is, SmBiT and LgBiT) to explore various protein-protein interactions, including amino\u2013amino (N\u2013N), amino\u2013carboxyl (N\u2013C) and carboxyl\u2013carboxyl (C\u2013C) interfaces. Specifically, 500\u2009ng of the receptor and 100\u2009ng of each \u03b2-arrestin construct were introduced into the cells. In the NanoBiT-BRET assay set-up, 500\u2009ng of the receptor and 1\u2009\u03bcg of location-specific tagged-mKO (including Cyto-mKO, CAAX-mKO, AP2-mKO\u00a0and 2xFYVE-mKO) were co-transfected with 200\u2009ng of each NanoBiT \u03b2-arrestin variant. Cyto-mKO and CAAX-mKO were used for the normalization of \u03b2-arrestin association studies (for example, N\u2013C interactions). For \u03b2-arrestin recruitment assays, cells received 500\u2009ng of receptor-LgBiT and 200\u2009ng of N-terminally tagged SmBiT-\u03b2-arrestin. To ascertain \u03b2-arrestin\u2019s recruitment to specific cellular locales, 500\u2009ng of the receptor and 1\u2009\u03bcg of either CAAX-LgBiT or 2xFYVE-LgBiT were used. Endocytosis assays were performed by co-transfecting 200\u2013500\u2009ng of receptor-RLuc with 11.5\u2009\u03bcg of 2xFYVE-Venus. For EKAR assays, 25\u2009ng of the biosensor was paired with 1\u2009\u03bcg of the receptor and 200\u2009ng of \u03b2-arrestin. For TRUPATH assay, 1:1:1:1\u2009\u03bcg of receptor:G\u03b1q:G\u03b2:G\u03b3 were used. On the following day (day 2 after transfection), cells underwent washing with PBS and detachment via trypsinization; they were then seeded onto a Corning Costar 96-well clear-bottomed, white-walled plate at a density of 70,000\u2013100,000 cells per well. The culture medium was replaced with clear minimal essential medium, enhanced with 2% foetal bovine serum, 1% penicillin\u2013streptomycin, 10\u2009mM HEPES, 1\u00d7 GlutaMAX and 1\u00d7 antibiotic\u2013antimycotic (Gibco). On day 3, the culture medium was removed, and cells were incubated with 80\u2009\u03bcl of 3\u2009\u03bcM coelenterazine h in Hanks\u2019\u00a0Balanced Salt Solution, further supplemented with 20\u2009mM HEPES buffer for 5\u2009min. Before ligand addition in split luciferase assays, three baseline reads were recorded to assess basal luminescence, which was then normalized to vehicle control conditions and shown as percent change in luminescence. Luminescence and BRET ratios were quantified using a BioTek Synergy Neo2 plate reader at 37\u2009\u00b0C. For BRET measurements, a 480\u2009nm wavelength filter for the donor and a 530\u2009nm or custom mKO 542\u2009nm long-pass emission filter for the acceptor were used. Net BRET was determined by subtracting the vehicle BRET ratio from the ligand-induced BRET ratio.<\/p>\n Confocal microscopy<\/p>\n HEK293T cells were seeded onto 35-mm dishes coated with poly-d-lysine and cultured until they reached 50\u201370% confluence. Following transfection using PEI, cells were incubated for an additional 16\u201324\u2009h to ensure adequate expression of the transfected constructs. Cells were then washed with PBS and serum-starved for 1\u2009h to synchronize cellular responses. Before imaging, cells were treated for 5\u2009min at 37\u2009\u00b0C with either 16HD or a control serum-free medium. After this pretreatment, cells were stimulated with ligands: 10\u2009\u03bcM isoproterenol, 1\u2009\u03bcM AngII or 1\u2009\u03bcM AVP. After stimulation, cells were fixed using 4% paraformaldehyde\u00a0(PFA) supplemented with Hoechst 33342 (1:1000, Thermo Fisher Scientific) for nuclear staining. Imaging was performed on a Zeiss 880 or 980 confocal microscope, using appropriate laser lines for Hoechst 33342 (400\u2009nm), GFP (480\u2009nm) and mKO (548\u2009nm).<\/p>\n Live cell microscopy<\/p>\n For live\u00a0cell imaging, HEK293T cells were similarly prepared and transfected as described for confocal microscopy. After the serum starvation period, cells were placed in a live\u00a0cell chamber system equipped with a temperature stage at 37\u2009\u00b0C. All live cell imaging was performed using 63\u00d7 objective. For optogenetic \u03b2-arrestin experiments, two laser wavelengths were used (488\u2009nm for Cry2 activation and 560\u2009nm for mCherry).<\/p>\n Fluorescence recovery after photobleaching<\/p>\n Fluorescence recovery after photobleaching was conducted using a Zeiss 980 confocal microscope with a 63\u00d7 objective, leveraging a 488\u2009nm laser for targeted bleaching of regions of interest. The procedure aimed to observe fluorescence recovery within these regions of interest over 3\u2009min at specified intervals. Small circular regions of interest were designated on either punctate structures or the diffuse cytosol, and bleaching was performed with the laser at 100% power to diminish fluorescence selectively. Following bleaching, fluorescence recovery was captured, allowing for the analysis of protein dynamics. Recovery data were processed using ImageJ for initial quantification. Microsoft Excel was then used to normalize the fluorescence intensity data, setting the five pre-bleach values to one for a standardized baseline and the immediate post-bleach intensity to 0.<\/p>\n Immunoblotting<\/p>\n Immunoblotting procedures were performed in accordance with previously established protocols. HEK293T cells were cultured in six-well plates and transiently transfected with \u03b2-arrestin pcDNA constructs using PEI. Following a 24-h post-transfection period, cells underwent serum starvation using minimum essential medium. Cells were then cooled on ice, rinsed with ice-cold PBS, and lysed using a buffer containing protease inhibitors Phos-STOP (Roche) and complete EDTA-free (Sigma). Lysates were agitated at 4\u2009\u00b0C for 45\u2009min and then centrifuged at more than 12,000g for 15\u2009min at 4\u2009\u00b0C to remove insoluble debris. The resulting supernatant was processed further. Protein samples were separated on SDS-10% polyacrylamide gels and transferred onto nitrocellulose membranes for blotting. Primary antibodies targeting phospho-ERK (1:1000 dilution, Cell Signalling Technology) and total ERK (1:1,000 dilution, Millipore Sigma) were applied overnight to evaluate ERK activation. The A1-CT antibody, specific for \u03b2-arrestin isoforms, and \u03b1-tubulin (Sigma-Aldrich) as a loading control were also used. Detection was facilitated by horseradish peroxidase-conjugated secondary antibodies (mouse anti-rabbit IgG or anti-mouse IgG) at a 1:3,000 dilution. The detection of immune complexes on the membranes was achieved using SuperSignal enhanced chemiluminescent substrate (Thermo Fisher) and documented with imaging equipment.<\/p>\n Imaging of \u03b2-arrestin puncta<\/p>\n On day one, HEK293T cells were transfected in six-well dishes and incubated for 24\u2009h. On day two 70\u2013100,000 cells were plated on 96-well poly-d-lysine-coated plates (Thermo Scientific). On day three, cells were fixed with 4% PFA. After 30\u2009min of fixation, cells were washed with 1\u00d7\u2009PBS for 10\u2009min \u00d7 3. For acquisition, Image Xpress Pico Automated Cell Imaging System (Molecular Devices) was used.<\/p>\n Quantification and statistical analysisQuantification of split\u00a0GFP-\u03b2-arrestin puncta<\/p>\n For analysis, Image Xpress Pico Automated Cell Imaging System (Molecular Devices) was used. Thresholds or size and image intensity were made to negative controls. Images were captured at 20\u00d7 and about 5,000 cells were analysed per well. Puncta were normalized to each cell with the nuclear marker.<\/p>\n Image analysis<\/p>\n Confocal Images were visualized using ImageJ. All image adjustments performed were identical and consistent. The line scan analysis function was used to measure the intensity of each channel.<\/p>\n Optogenetic puncta quantification<\/p>\n Condensates were quantified on a per cell basis on a single optical plane using the \u2018Surfaces\u2019 module in Imaris (Bitplane; v.10.2). Cell outlines were determined from the first 10\u2009s of the time-lapse, before condensate formation. Condensate detection thresholds were determined on a per-cell basis using the mean intensity of the cell measured at the start of each time-lapse. The remaining surface detection parameters were held constant across cells and conditions (smoothing applied\u2009=\u2009true, surface grain size\u2009=\u20090.07\u2009\u03bcm, largest sphere diameter\u2009=\u20093\u2009\u03bcm). For each cell, the total number of condensates were quantified at 1\u2009s intervals over a 120\u2009s imaging period. Two to four cells were quantified across multiple independent images for an average of nine cells per experimental condition. For quantification, only cells with similar expression were used for quantification.<\/p>\n Line scan analysis<\/p>\n Spatial localization of \u03b2-arrestin and GPCRs were quantified using line scan analysis in ImageJ (National Institutes of Health; v.1.54f). For each condition, a straight\u00a0line segment (18\u201320\u2009\u03bcm) was drawn manually using the line tool such that each line extended from one side of the cell membrane to the opposite side, spanning the full width of the cell. Cell membrane was determined using the GPCR channel. Fluorescence intensity profiles for the \u03b2-arrestin and GPCR channels across the entire line segment were extracted using the plot profile function.<\/p>\n Statistics and reproducibility<\/p>\n Statistical methods were not used to predetermine sample size. Blinding and randomization were not used.\u00a0Data were analysed in Microsoft Excel and graphed in GraphPad Prism v.11.0 (GraphPad). Dose-response curves were fitted to log agonist versus stimulus with three parameters with the minimum baseline corrected to zero. Statistical tests were performed using a two-way ANOVA when comparing different \u03b2-arrestin mutants in time response assays AUC. Further details of statistical analysis and replicates can be found in the figure legend. Crucial plate-based experiments were independently replicated by at least two different investigators whenever feasible. Specific P\u00a0values for Figs. 1<\/a>\u20135<\/a> can be found in Supplementary Table 2<\/a>.<\/p>\n Materials availability<\/p>\n All plasmids generated in this study will be distributed on reasonable request.<\/p>\n Experimental model and subject details<\/p>\n All cell lines are periodicially tested for mycoplasma commnication.\u00a0HEK 293T cells, including a \u03b2-arrestin 1\u20132 KO variant, were cultured in Dulbecco\u2019s modification of Eagle\u2019s medium with 10% foetal bovine serum and 1% antibiotic\u2013antimycotic solution from Gibco, under conditions of 37\u2009\u00b0C and 5% CO2 humidity. The \u03b2-arrestin 1\u20132 KO cells, created through CRISPR\u2013Cas9 genome editing, were authenticated by immunoblot analysis and obtained from A. Inoue.<\/p>\n For experiments requiring confocal microscopy, both HEK293T and \u03b2-arrestin 1\u20132 KO cells were seeded on 35-mm glass-bottomed dishes pre-coated with either poly-d-lysine or rat tail collagen, aiming for a confluence between 40% and\u00a070%. In the case of 96-well plate assays, cell density was adjusted to 70,000\u2013100,000 HEK293T cells per well.<\/p>\n Transient transfections were performed using OPTI-MEM and PEI at a PEI-to-DNA mass ratio of 3:1. Cells designated for confocal microscopy analysis were prepared and imaged after 16\u201324\u2009h post-transfection, adhering to the same timeline for BRET and split nanoluciferase assays.<\/p>\n Reporting summary<\/p>\n