p110δ regulates fission of TGN-derived, TNF-containing tubular carriers. (A–C) RAW264.7 macrophages coexpressing YFP-p230GRIP (green) and TNF-mCherry (red) were treated with LPS only (A; control), LPS and 15 nM wortmannin (B), or LPS and 5 µM IC87114 (C) for at least 30 min before live cell imaging. (A) Selected stills from Video 2 depict tubular carriers labeled for YFP-p230GRIP emerging from the TGN and rapidly undergoing fission (white and green arrows) to release boluses of TNF-mCherry as cargo (dotted circles in grayscale insets). (B and C) Stills from Videos 3 and 4, respectively, also show YFP-p230GRIP tubules loaded with TNF-mCherry that were formed but cannot undergo fission. (D) Counting of fission events in TNF-mCherry–loaded YFP-p230GRIP tubules in live cells treated with LPS only (control), LPS and wortmannin (wortm), or LPS and IC87114 (IC87114). 30–40 cells were analyzed over eight transfections for each treatment. Error bars indicate mean ± SEM. **, P = 0.0022; ***, P < 0.0001. Regions of colocalization or interest are indicated by boxes and are magnified in insets. Bars, 20 µm.
