Live-cell TIRF microscopy

Cells at ∼50% confluence, expressing the gene of interest, were seeded on a 35-mm glass-bottom dish (Cellvis) coated with concanavalin A for 30–60 min. Before imaging, the total volume was brought up to 2 ml with fresh Schneider’s medium containing FBS. Multicolor, live-cell TIRF videos of all the constructs (EGFP-α-tubulin and γ-tubulin-Tag-RFP-T; EGFP-α-tubulin, γ-tubulin-Tag-RFP-T, and mTurquoise2-Dgt5; Tag-RFP-T-α-tubulin and Rhotekin-EGFP) were acquired on a Nikon Ti-E microscope equipped with a 100× 1.49-NA differential interference contrast Apochromat oil-immersion objective, a Hamamatsu ORCA-Flash 4.0 LT digital complementary metal-oxide semiconductor camera ({"type":"entrez-nucleotide","attrs":{"text":"C11440","term_id":"1536511","term_text":"C11440"}}C11440), four laser lines (447, 488, 561, and 641 nm), and MetaMorph software (Molecular Devices). Metamorph was used to control the imaging systems.