FIGURE 2.

IL-33 displays dynamic nucleo-cytoplasmic flux. A, tetracysteine-tagged IL-33 (TC-IL-33) displays nuclear and cytoplasmic localization. A fibroblast cell line stably expressing TC-IL-33 was probed with tetracysteine-avid fluorescein derivative and one of two anti-IL-33 antibodies (commercially available N1 or a11 raised by our laboratory). Confocal microscopy revealed nuclear (arrowheads) and cytoplasmic (arrows) localization of TC-IL-33 (pseudocolored green), similar to the pattern of wild-type IL-33 staining in mock-transfected cells (anti-IL33 antibody signal pseudocolored red). Note mock-transfected cells did not evidence fluorescein fluorescence. Scale bars represent 10 μm. B, cytoplasmic IL-33 is housed within vesicles. As the tetracysteine-avid resorufin derivative can render 3,3′-diaminobenzidine electron dense,(8) the subcellular localization of TC-IL-33 was interrogated by transmission electron microscopy. Nuclei displayed punctate electron densities (arrowheads) concentrated in areas of euchromatin (Ec). The cytoplasm evidenced electron densities within membrane-bound structures (arrows). P and N denote plasma and nuclear membranes, respectively. Hc indicates heterochromatin. Scale bars represent 1 μm. C, IL-33 displays dynamic nucleo-cytoplasmic flux. Fibroblasts stably expressing TC-IL-33 were pulsed with tetracysteine-avid fluorescein derivative and imaged by time-lapse epifluorescent microscopy. Shown are representative images at 0 and 80 min post-pulse. Over the chase period, a transposition of fluorescence from the nucleus (arrowheads) to the cytoplasmic space (arrows) is evident. A time-lapse movie may be viewed as part of the supplemental materials. Scale bars represent 10 μm. D, newly synthesized IL-33 molecules are nuclear in their localization. Pulse-chase-pulse experiments were performed by exposing fibroblasts stably expressing TC-IL-33 to a primary application of tetracysteine-avid fluorescein derivative followed by secondary resorufin derivative exposure after a variable chase period. By this method, all existent IL-33 molecules at the time of the initial pulse were rendered fluorescein-positive. Molecules synthesized after the primary pulse were non-fluorescent until application of the resorufin derivative at the end of the chase period, allowing differential fluorescence of molecules synthesized at different times. Cells treated with resorufin derivative without a chase period did not evidence red fluorescence in any subcellular compartment. Cells permitted a 40-min chase period evidenced fluorescein fluorescence in the cytoplasm and resorufin fluorescence in the nucleus (arrowhead). Scale bars represent 10 μm.