Cell culture and generation of stable Dynamin chimera cell lines

The normal human retinal pigmental epithelial-19 (herein referred to as ARPE) cell line was acquired from ATCC and cultured in DMEM/F12 (1:1) (Life Technologies 11330-032) supplemented with 10% fetal bovine serum (FBS; Sigma F0926). ARPE19 cells were infected with pLVX-Puro-mRuby2-CLCa lentiviral expression vector (Srinivasan, Burckhardt, et al., 2018 ) followed by FACS sorting to select mRuby–CLC-expressing cells. Incorporation of mRuby-CLCa in complex with clathrin heavy chain was confirmed by immunoprecipitation of clathrin heavy chain and analyzing the percentage of mRuby-CLCa pulled down with clathrin heavy chain. ARPE mRuby-CLCa cells were then used to generate stable dynamin chimera cell lines. All the dynamin chimeras (Supplemental Table S2) were cloned in a retroviral expression vector pMIEG3 using seamless cloning. The seamless cloning is a method that uses PCR to amplify the insert. The insert and the vector fragment are then treated with exonuclease to create overhangs. This is followed by treatment with a ligase or in vivo repair in bacteria to allow the joining of insert and vector. Such cloning method is scarless and sequence independent compared with the restriction enzyme-based traditional cut–paste method. pMIEG3 is a modified form of pMIG vector (Williams et al., 2000 ) with enhanced GFP fluorescence. For the purpose of this study, the pMIEG3 backbone was modified to insert an N-terminal HA tag and the IRES was removed to make C-terminal GFP fusions of dynamin chimeras. To achieve this, dynamin chimera vectors were PCR amplified using the primers listed in Supplemental Table S2 so as to exclude the IRES region and generate a PCR product with homologous overhanging regions to allow for subsequent homologous recombination when transformed in competent bacterial cells. The removal of IRES was confirmed by sequencing. ARPE CLCa-mRuby cells were transduced with retroviral dynamin chimera constructs and the cells were FACS sorted to select GFP-positive cells, while maintaining CLCa-mRuby expression. The constitutively active Dyn1 chimera plasmids were generated by site-directed mutagenesis of wild-type chimeras to replace serine at positions 774 and 778 to alanine (here onward referred to as AA chimeras) using primers listed in Supplemental Table 2 and confirmed by sequencing. Expression levels of chimera were checked by Western blotting using pan-dynamin antibody 4003 (in-house Rabbit).