Effect of C3 exoenzyme treatment on Citron-K localization during cell division. Recombinant C3 exoenzyme was introduced into HeLa cells enriched in the S-phase by electroporation, and its effects on cell division and localization of endogenous Citron-K were examined. (A) Failure of cytokinesis in HeLa cells treated with C3 exoenzyme. Almost all the treated cells became binucleate 16 hours after electroporation as shown by DAPI staining (blue). Microtubules are stained in green. (B) Effect of C3 exoenzyme treatment on Citron-K localization during cell division. HeLa cells without (left-hand pairs of panels) or with (middle and right-hand pairs of panels) C3 exoenzyme treatment were fixed in various phases of cell division and stained with anti-Citron antibody (green). Microtubules and DNA were stained with anti-β-tubulin antibody (red) and TOPRO3 (blue), respectively. The left panels of each pair represent merged images. Note that Rho inactivation by C3 exoenzyme treatment did not affect Citron-K localization in prometa- and metaphase, but prevented the transfer of Citron-K to the cortex in telophase, which instead was associated with the spindle midzone (middle bottom pairs of panels). The Citron-K signal in the spindle midzone was abolished in the presence of the antigenic peptide (the right bottom pair of panels). Bars, 10 μm.

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-β-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.

Colocalization of RhoA and Citron-K in cleavage furrow of HeLa cells. Colocalization of Rho and Citron-K was examined by staining endogenous Rho with anti-Rho antibody (red, right-hand panels) in HeLa cells expressing GFP-Citron-K (green, middle panels) in various stages of cytokinesis. Left-hand panels represent merged images. In interphase, Rho shows homogenous staining in the cytoplasm, whereas Citron-K shows particulate signals (A). Punctate signals for Rho in the nucleus appear nonspecific, because such signals are only found by this polyclonal antibody 119 used in this experiment, and not by the other monoclonal antibody 26C4. GFP-Citron-K and endogenous RhoA colocalize around the cell equator of cells in the early stage (B), in the ingressing cleavage furrow in the middle stage (C), and are concentrated together at the cleavage site in the end stage (D) of cytokinesis. In addition, a portion of GFP-Citron-K remains as aggregates in the cytoplasm, where no colocalization with Rho was observed (for example, see arrowheads in B and C). Confocal sections of each cell are shown. Note that Citron-K and a part of Rho also colocalize in the midbody of post mitotic cells (E). Blue in E is β-tubulin. Bars, 10 μm.

Translocation of GFP-Citron-K to the cortex of interphase cells by expression of dominant active Rho. (A) Effects of expression of dominant active Rho on localization of Citron-K. Myc-Citron-K and GFP-Val14RhoA were co-expressed in HeLa cells. Citron-K (red) and Val14RhoA (green) are colocalized in band-like structures at the bottom of cells (bottom slice), whereas in the middle of the cell, Citron-K is present as an aggregate without colocalization with Val14Rho (the middle slice, arrowheads). Band-like structure of Citron-K and Val14RhoA at the bottom consists of small patches. (B) Vertical views of GFP-Citron-K localization in HeLa cells co-expressing Myc-Val14RhoA and GFP-Citron-K. Note that expression of Val14RhoA (red) disintegrates cytoplasmic aggregates of GFP-Citron-K (green) and translocates it to the cell cortex to form band-like structures. Signals of myc-Val14RhoA and GFP-Citron-K appear not to colocalize completely because the fluorescence of Texas Red-Myc-Val14RhoA is not so strong as GFP-Citron-K, and only a small portion of expressed Rho colocalize with Citron-K. (C) Effects of expression of GFP-tagged wild-type RhoA (the left pair of panels) and GFP-tagged Asn19RhoA (the right-hand pair of panels) on localization of Myc-tagged Citron-K. Myc-Citron-K (red) remains as aggregates when wild-type or dominant negative RhoA (green) is co-expressed. (D) Effects of Rac expression on Citron-K localization. GFP-Citron-K (green) and either Myc-tagged Val12Rac1 (red) (the left pair of panels) or FLAG-tagged Asn17Rac1 (red) (the right pair of panels) were co-expressed in HeLa cells. Note that either expression did not affect the cytoplasmic aggregates of GFP-Citron-K. (E) Effects of Cdc42 expression. GFP-Citron-K (green) and either Myc-tagged Val12Cdc42 (red) (the left pair of panels) or Myc-tagged Asn17Cdc42 (red) (the right pair of panels) were co-expressed in HeLa cells. Note that either expression did not affect the cytoplasmic aggregates of GFP-Citron-K. C, D and E all show the bottom slices of interphase cells. Bars, 10 μm.

Mutual exclusion of Citron-K-containing band-like structures and actin stress fibers. GFP-Citron-K and Myc-Val14RhoA were co-transfected into HeLa cells. Actin stress fibers (red) and band-like structures of Citron-K (green) exist on the same plane at the bottom of interphase cells, but the signals do not overlap at all (a merged image, left). Bar, 10 μm.

Mutual exclusion of Citron-K-containing structures and actin cytoskeleton in the equatorial cell cortex during cytokinesis. (A and B) Localization of endogenous Citron-K and F-actin. Two HeLa cells at different stages of cytokinesis were chosen, and stained for endogenous Citron-K (green), F-actin (red), and DNA (blue). Citron-K and F-actin appear to overlap in the very early stage of cytokinesis (A), but are clearly separated in the late stage of cytokinesis (B). (C,D) Localization of GFP-Citron-K (green) and F-actin (red). GFP-Citron-K-expressing HeLa cells undergoing ingression of the cleavage furrow (C) and that at the end stage of cytokinesis (D) were chosen. In C, several confocal sections encompassing the cleavage furrow of a dividing cell are piled up and shown. β-Tubulin was stained blue in D. Note that GFP-Citron-K and F-actin do not colocalize. Scale bars, 10 μm.