Generation of Constructs
Constructs were created using a modified technique for overlap cloning. Flexible linkers made of glycine-serine repeats (GGGGS), varying in length from 18 to 33 amino acids, were added between coding sequences for fluorescent proteins or luciferases and the receptors, transducers, biosensors, or adaptor proteins. The mutants of SmBiT-β-arrestin IP6, AP2, and clathrin were made using a previously described overlap cloning method with SmBiT. To create the 2xFYVE-mKO construct, Cyto-mKO was integrated into the 2xFYVE-LgBiT vector through overlap cloning.
Generation of β-arrestin-split GFP
The β-arrestin-split GFP constructs stemmed from pcDNA3.1-GFP(1–10) and pEGFP-GFP11-Actin, then cloned into either SmBiT-β-arrestin or β-arrestin-SmBiT.
Generation of Stable Cell Lines
Stable β-arrestin knock-in cell lines were generated by Cyagen. Briefly, the Arrb1–2 (C-terminal linker-GFP11) knock-in HEK293T cells were cultured in six-well plates for 24 hours. Cas9 nuclease and guide RNAs were mixed to form ribonucleoprotein complexes, and the donor vector was electroporated into the target cells. After 24 to 48 hours post-electroporation, the cell pool was sorted and evaluated using DAPI-negative staining. High-editing-efficiency cell pools were chosen for monoclonal preparation, where single cells were placed in 96-well plates through limiting dilution. Clones that grew well were expanded and analyzed through PCR and sequencing to ensure accurate C-terminal linker-GFP11 integration.
Mutagenesis of β-arrestin IP6 and IDR Mutants
Mutations were created using the QuikChange site-directed mutagenesis kit. The resulting mutations included ΔIP6-N, ΔIP6-C, ΔIP6-N–C, ΔIP6-NT, ΔIP6-NT–C, ΔIDR, and ΔCT.
Opto-β-arrestin Constructs
Opto-β-arrestin constructs were based on earlier work. pHR-mCh-Cry2WT and pHR-FUSN-mch-Cry2WT were cloned into the pcDNA backbone, followed by overlap cloning to generate all opto-β-arrestin constructs.
Immunofluorescence
For immunofluorescence, HEK293T cells were fixed with 4% PFA for 20 minutes and washed three times with phosphate-buffered saline (PBS). The cells were then incubated in donkey serum blocking buffer with Triton X-100 and washed again. Primary antibodies targeting β-arrestin 1 or β-arrestin 2 were applied overnight at 4°C. Afterward, cells were washed and treated with secondary antibodies and Hoechst 33342 before further washing.
BRET and Split Nanoluciferase Assays
In the BRET and split luciferase assays, HEK293T cells were cultured and transiently transfected using PEI. β-arrestin association was evaluated using split NanoLuc components to study various protein-protein interactions. Receptors and β-arrestin constructs were introduced into cells in specified quantities. Following transfection, cells underwent washing and reseeding onto 96-well plates. After 48 hours, luminescence measurements were taken using specialized filters, with net BRET calculated by adjusting for vehicle control conditions.
Confocal Microscopy
In confocal microscopy, HEK293T cells were prepared and cultured to 50-70% confluence before imaging. Following transfection and a serum starvation period, cells were stimulated with various ligands, fixed, and imaged using appropriate settings on a confocal microscope.
Live Cell Microscopy
For live cell imaging, HEK293T cells underwent a similar preparation process. Cells were maintained at 37°C in a live cell chamber during imaging, employing two different laser wavelengths for activation purposes.
Fluorescence Recovery After Photobleaching
Using a confocal microscope, fluorescence recovery after photobleaching was assessed by targeting specific regions and monitoring recovery over time. Data generated were processed for initial quantification and normalized.
Immunoblotting
Immunoblotting followed established protocols where HEK293T cells were cultured, transfected, and lysed. Proteins were separated via SDS-PAGE and transferred to membranes for antibody detection of targeted proteins, with imaging performed for results analysis.
Imaging of β-arrestin Puncta
For imaging β-arrestin puncta, HEK293T cells were transfected and plated in poly-D-lysine-coated plates. After fixation, imaging was conducted using an automated cell imaging system.
Quantification and Statistical Analysis
Quantification of Split GFP-β-arrestin Puncta
Quantification utilized an automated imaging system, analyzing normalization and image intensity across samples.
Image Analysis
Confocal images were processed using ImageJ for consistent adjustments and intensity measurements.
Optogenetic Puncta Quantification
Condensates were quantified across multiple cells using software, measuring formation across time intervals.
Line Scan Analysis
Spatial localization of proteins was analyze through line scan analysis, assessing fluorescence intensity profiles.
Statistics and Reproducibility
Statistical methods were not predetermined, and data analyses were completed using Excel and appropriate graphing software. Dose-response curves were fitted appropriately, and independent replication was conducted where necessary.
Materials Availability
All plasmids created in this study will be available upon reasonable request.
Experimental Model and Subject Details
All cell lines are regularly checked for mycoplasma. HEK 293T cells, including a β-arrestin 1-2 KO variant, were grown in specified conditions. The β-arrestin KO cells were authenticated and utilized per experimental needs. Transient transfections were achieved using specific methods.
Reporting Summary
More details on the research design are accessible through supplementary materials linked to the article.
