GoKawiilGeneration of constructs
Constructs were developed using a modified overlap cloning technique60. Flexible linkers, consisting of glycine–serine 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-β-arrestin IP 6 , 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.
Generation of β-arrestin-split GFP
β-Arrestin-split GFP constructs were derived from pcDNA3.1-GFP(1–10) (Addgene no. 70219) and pEGFP-GFP11-Actin (Addgene no. 181966) and cloned into either SmBiT-β-arrestin or β-arrestin-SmBiT.
Generation of stable cell lines
β-Arrestin knock-in cell lines were made by Cyagen. In short, the Arrb1–2 (C terminal linker-GFP11) knock-in HEK293T cells wereseeded into six-well plates and cultured for 24 h. Cas9 nuclease (0.2 nmol) and guide RNAs (0.1 nmol; sequences in Supplementary Table 1) were pre-incubated to assemble ribonucleoprotein complexes, and 0.5 nmol of donor vector was electroporated into the target cells. Within 24–48 h 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) and sequencing (Genscript) to confirm precise C terminal-linker-GFP11 integration.
Mutagenesis of β-arrestin IP 6 and IDR mutants
Mutation generation was performed using the QuikChange site-directed mutagenesis kit (Agilent). The mutations produced included ΔIP 6 -N, ΔIP 6 -C, ΔIP 6 -N–C, ΔIP 6 -NT, ΔIP 6 -NT–C, ΔIDR and ΔCT.
Opto-β-arrestin constructs
Opto-β-arrestin constructs were designed on the basis of those from a past work35. 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-β-arrestin constructs.
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