Engineered HeLa Cell Lines for Dyn2 and EHD2 Studies

The HeLa Flp-In T-REx Dyn2-EGFP-P2A-Caveolin1-mCherry, EGFP-EHD2-P2A-Caveolin1-mCherry, and Pac2-EGFP-P2A-Caveolin1-mCherry constructs were generated by linearizing pcDNA/FRT/TO/Caveolin1-mCherry (Hubert et al., 2020b (link)) with the restriction enzyme HindIII (Thermo Fisher Scientific). The DNA encoding the EGFP-fusion proteins and the P2A peptide was amplified by PCR and inserted by Gibson assembly using NEBuilder HiFi DNA assembly master mix (New England BioLabs). For creating flanking insert fragments, the following primers were used for Dyn2-EGFP-P2A: 5′-GAC​TCT​AGC​GTT​TAA​ACT​TAA​TGG​GCA​ACC​GCG​GGA​TG-3′ paired with 5′-TTC​CAT​CGA​TCT​TGT​ACA​GCT​CGT​CCA​TGC​C-3′ and 5′-TGG​ACG​AGC​TGT​ACA​AGA​TCG​ATG​GAA​GCG​GAG​CTA​C-3′ paired with 5′-TGG​ATC​CGA​GCT​CGG​TAC​CAC​CTC​TAG​GTC​CAG​GGT​TC-3′. Primers used for Pac2-EGFP-P2A were: 5′-GAC​TCT​AGC​GTT​TAA​ACT​TAA​T GTC​TGT​CAC​CTA​CGA​TG-3′ paired with 5′-TTC​CAT​CGA​TCT​TGT​ACA​GCT​CGT​CCA​TGC​C-3′ and 5′-TGG​ACG​AGC​TGT​ACA​AGA​TCG​ATG​GAA​GCG​GAG​CTA​C-3′ paired with 5′-TGG​ATC​CGA​GCT​CGG​TAC​CAC​CTC​TAG​GTC​CAG​GGT​TC-3′. For creating flanking insert fragments, the following primers were used for EGFP-EHD2-P2A: 5′-GAC​TCT​AGC​GTT​TAA​ACT​TAA​TGG​TGA​GCA​AGG​GCG​AG-3′ paired with 5′-TTC​CAT​CGA​TTT​CAG​CAG​AGC​CCT​TCT​G-3′ and 5′-CTC​TGC​TGA​AAT​CGA​TGG​AAG​CGG​AGC​TAC-3′ paired with 5′-GGA​TCC​GAG​CTC​GGT​ACC-3′. The HeLa Flp-In T-REx K44A Dyn2-EGFP-P2A-Cav1-mCherry construct was obtained by in vitro mutagenesis of the pcDNA/FRT/TO/Dyn2-EGFP-P2A-Cav1-mCherry construct exchanging lysine at position 44 to an alanine using these primers: 5′-CCA​GAG​CGC​CGG​CGCGAG​TTC​GGT​GCT​C-3′ and 5′-GAG​CAC​CGA​ACT​CGCGCC​GGC​GCT​CTG​G-3′. For creation of the HeLa Flp-In T-REx I684K Dyn2-EGFP-P2A-Cav1-mCherry construct the following primers were used to exchange isoleucine at position 684 to a lysine: 5′-GAC​CAT​CAT​GCA​CCT​CAT​GAAGAAC​AAC​ACA​AAG​GCT​TC-3′ and 5′-GAA​GGC​CTT​TGT​GTT​GTTCTTCA​TGA​GGT​GCA​TGA​TGG​TC-3′. The underlining represents the nucleotides in the primer correspond to specific mutations. The Flp-In TRex HeLa cell lines were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 μg/ml hygromycin B (Thermo Fisher Scientific), and 5 μg/ml blasticidin S HCl (Thermo Fisher Scientific) for plasmid selection at 37°C, 5% CO₂. Expression at near endogenous levels was induced by incubation with 0.5 ng/ml (Cav1-mCh) and 1.0 ng/ml (EGFP-fusion-P2A-Cav1mCh) doxycycline hyclate (Dox; Sigma-Aldrich) for 16–24 h. All cell lines tested negative for mycoplasma. For generation of the ΔNΔEH EHD2-BFP expression vector, the ΔNΔEH EHD2 mRNA was subcloned from ΔNΔEH EHD2-mCherry construct (Hoernke et al., 2017 (link)) using restriction enzymes XhoI and BamHI (Thermo Fisher Scientific) and inserted into the pTagBFP-C (Evrogen) expression vector.