Video 4. Example of a FMNL1 KO/LifeAct-GFP T cell failing polymerize actin at the back of the cell and unable to migrate through a microchannel constriction.

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FMNL1 KO/LifeAct-GFP T cells were activated ex-vivo, labeled with Hoechst to visualize their nuclei, and added to microchannel devices. T cells crawling in the microchannels were imaged at 37°C for 2–6 hr. Brightfield and fluorescent images were acquired every 30 s. Top images, LifeAct-GFP (green) and Hoechst (DNA, blue) overlaid on the brightfield channel (gray). Bottom images, pseudocolor rendition of the LifeAct-GFP channel. This representative FMNL1 KO/LifeAct-GFP T cell lacks posterior perinuclear actin polymerization as its nucleus engages the microchannel constriction and is unable to migrate through the constriction. Time is displayed as min:s.