FIGURE 3.

Nucleo-cytoplasmic shift of IL-33 is dependent on the nuclear pore complex. A, IL-33 localizes to nuclear pore complexes. High-magnification transmission electron microscopy of fibroblasts stably expressing TC-IL-33 exposed to the tetracysteine-avid resorufin-derivative displayed electron densities highlighting nuclear pore complexes (NPC). The left panel shows the complex in cross section. The middle panel depicts the complex en face whereby IL-33 molecules can be seen within the pore channel itself (enlarged in right panel). In these images, IL-33 can also be seen localizing to a chromatin strand (Cr). P and N denote plasma and nuclear membranes respectively. Scale bars represent 1 μm. B, normal nuclear pore complex function is obligatory for nuclear efflux of IL-33. Fibroblasts stably expressing TC-IL-33 were microinjected with either Texas-red conjugated wheat germ agglutinin (WGA) to block nuclear pore molecular transit or Texas-red conjugated dextran (Dx) as control. Cells were then treated with the tetracysteine-avid fluorescein derivative and allowed a 60-min chase period. In the left panel, Texas-red wheat germ agglutinin can be seen binding the plasma and nuclear membranes (pseudocolored red). Clear retention of nuclear fluorescein-labeled IL-33 can be seen (pseudocolored green, arrowhead). In contrast, control cells injected with Texas-red dextran (right panel) evidence efflux of IL-33 from the nucleus into the cytoplasm (arrow). Scale bars represent 5 μm.