FIGURE 4.

IL-33 translocation depends on an intact microtubule network, and is ATP dependent. A–C, kinetics of IL-33 nucleo-cytoplasmic flux is enhanced upon microtubule disruption. A fibroblast cell line stably expressing TC-IL-33 was pulsed with tetracysteine-avid fluorescein derivative and allowed a chase period of 0 or 60 min (left and right panels, respectively) under variable conditions of chemical cytoskeletal disruption. A denotes control condition. B depicts experiments conducted in the presence of latrunculin B, to disrupt the actin cytoskeleton. C depicts experiments conducted in the presence of nocodazole, to disrupt the microtubule network. Under conditions of microtubule disruption, cytoplasmic vesicular IL-33 can be seen in the absence of a chase period (arrowhead). D, cytoplasmic transit of vesicular IL-33 is ATP dependent. Tetracysteine-avid fluorescein derivative pulse-chase was conducted under conditions of cellular ATP depletion (glucose free DMEM supplemented with 2-deoxy-d-glucose and 6 mm sodium azide). At the end of a 60-min chase period, punctate fluorescein staining can be seen relegated to a perinuclear position (arrowhead) rather than distributed throughout the cytoplasm as is seen in the control condition (panel A). Fluorescein staining is depicted in green. Phalloidin-based actin (latrunculin and ATP-depletion experiments) or anti-tubulin (nocodazole experiments) staining is depicted in red. Scale bars represent 10 μm.