Fig. 3. Localization of GFP-tagged Citron-K during cell cycle. HeLa cells expressing GFP-Citron-K in various phases of cell cycle were fixed. Expressed GFP-Citron-K was localized by GFP fluorescence, and microtubules and DNA were stained with anti-ß-tubulin antibody (red) and TOPRO3 (blue), respectively. Note that GFP-Citron-K is again detected as punctate cytoplasmic signals in interphase cells (arrowheads in a), disperses in the cytoplasm in prometa- and metaphase (b and c), accumulate in the cortex of the cleavage furrow in ana- to telophase (d) and is present in the midbody after division (e and f). In about 50% of metaphase cells overexpressing Citron-K, the GFP-Citron-K aggregates appeared to attach to the cell cortex as shown in c. (B) Electron microscopy of GFP-Citron-K aggregates in interphase. Punctate fluorescence signals of GFP-Citron-K expressed in HeLa cells were identified first in a live cell by fluorescence microscopy and subjected to electron microscopy. Note that GFP-Citron-K is seen as an amorphous structure with many holes, which is not apparently enclosed by lipid bilayers. (C) Effect of C3 exoenzyme treatment on GFP-Citron-K localization in telophase. C3 exoenzyme was introduced into HeLa cells expressing GFP-Citron-K (green) by electroporation, and Citron-K localization in telophase was examined. Note that GFP signals were associated with the spindle midzone. The left-hand panels are merged images with microtubules stained in red and DNA stained in blue. Bars, 10 µm except in the right-hand panel of B.