Movie 1

MICAL-L1 controls TNF traffic between Golgi and plasma membrane. HeLa stable cell lines expressing Scramble and 2 different shRNA fused to GFP targeting human MICAL-L1 were transfected with TNFα-SBP-mCherry using the RUSH system. Cells were then imaged using a spinning disk confocal microscope at 37 °C. Before biotin treatment, we detected TNFα-SBP-mCherry as scattered dots corresponding to ER exit sites. After biotin addition, TNFα-SBP-mCherry was observed in the Golgi complex within 10 min. After 15 min, post-Golgi vesicles appeared and plasma membrane staining became visible whereas signal at the crossed the perinuclear region decreased disappearing almost completely after 40 min. TNFα proteins crossed the perinuclear region in 10–15 min on average in control cells whereas in MICAL-L1 depleted cells, TNFα cargoes were delayed and crossed the crossed the perinuclear region in 30 min on average. Movies are presented as single color movies of TNFα-SBP-mCherry.