Cath.-a-differentiated (CAD) cells were purchased from Sigma-Aldrich and cultured in DMEM/F12 medium (Gibco) supplemented with 8% fetal calf serum, 1% L-Glutamine, and 1% penicillin-streptomycin. Two to four hours prior to imaging, CAD cells were plated on coverslips coated overnight at 4 °C with 10 µg/mL laminin (Sigma-Aldrich). DMEM/F12 medium without phenol red (Gibco) supplemented with 15mM HEPES was used for live-cell imaging. Cell lines were also routinely tested for mycoplasma using the Universal Detection Kit (ATCC). PFN1 KO cells were generated with CRISPR/Cas9 as previously described (28 (link)). CAD cells were transfected with plasmid DNA via electroporation as previously described (28 (link)) or with LipoD293 (SignaGen, “Hard-To-Transfect Mammalian Cell” protocol). Cells expressing HaloTag constructs were incubated overnight with 10–100 nM Janelia Fluor 646 HaloTag ligand (Promega) (61 (link)). EGFP-NM2A knock-in MEFs were generated from mice (62 (link)), and isolated and cultured as previously described (40 (link)).