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Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis.

Tatsumoto T, Xie X, Blumenthal R, Okamoto I, Miki T - J. Cell Biol. (1999)

Bottom Line: Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis.Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis.These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

View Article: PubMed Central - PubMed

Affiliation: Molecular Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.

ABSTRACT
Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

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Effects of a dominant negative mutant of ECT2 on cytokinesis. (A) Subcellular localization of exogenously expressed ECT2 protein. The full-length (ECT2-F; panels a and b), NH2-terminal half (ECT2-N; panels c–f), or COOH-terminal half (ECT2-C; panels g and h) of ECT2 was transiently expressed in U2OS cells as a GFP-fused protein. GFP fusion proteins were detected by green fluorescence (panels a, c, e, and g). DNA was stained with DAPI (panels b, d, f, and h). (B) The NH2-terminal domain of ECT2 acts as a dominant negative mutant for cytokinesis. Cells were transfected with GFP-fused ECT2-F, ECT2-N, or ECT2-C, or GFP vector alone. GFP-expressed cells are visualized by green fluorescence (panels a and c). DNA was stained with DAPI (panels b and d). Arrowheads indicate multinucleated cells. Bars, 20 μm. (Right panel) GFP-expressing multinucleated cells were scored under immunofluorescent microscopy 72 h after transfection. Data are average of three independent experiments.
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Figure 3: Effects of a dominant negative mutant of ECT2 on cytokinesis. (A) Subcellular localization of exogenously expressed ECT2 protein. The full-length (ECT2-F; panels a and b), NH2-terminal half (ECT2-N; panels c–f), or COOH-terminal half (ECT2-C; panels g and h) of ECT2 was transiently expressed in U2OS cells as a GFP-fused protein. GFP fusion proteins were detected by green fluorescence (panels a, c, e, and g). DNA was stained with DAPI (panels b, d, f, and h). (B) The NH2-terminal domain of ECT2 acts as a dominant negative mutant for cytokinesis. Cells were transfected with GFP-fused ECT2-F, ECT2-N, or ECT2-C, or GFP vector alone. GFP-expressed cells are visualized by green fluorescence (panels a and c). DNA was stained with DAPI (panels b and d). Arrowheads indicate multinucleated cells. Bars, 20 μm. (Right panel) GFP-expressing multinucleated cells were scored under immunofluorescent microscopy 72 h after transfection. Data are average of three independent experiments.

Mentions: Next, we examined the subcellular localization of endogenous ECT2 during cell cycle progression by immunofluorescent analysis of HeLa cells. Interestingly, in interphase cells ECT2 was located predominantly in the nucleus, where no expression of Rho family proteins has been reported (Fig. 2 a). After nuclear membrane breakdown in prometaphase, ECT2 spread into the cytoplasm (Fig. 2 b). During metaphase, ECT2 accumulated in the regions where the mitotic spindle is present (Fig. 2 c). Costaining of ECT2 and tubulin generated the yellow colocalization signal. As cells entered anaphase, ECT2 appeared to concentrate in the central region of the spindle (Fig. 2 d). In late anaphase and telophase, ECT2 was mainly located in the midzone, where the cleavage furrow is formed (Fig. 2 e). During cytokinesis, ECT2 accumulated at the midbody, a region in the middle of the bridge that connects the two daughter cells (Fig. 2 f). When the cells exited mitosis and the nuclear envelope reassembled, ECT2 translocated to the nucleus again (data not shown). To locate the region of ECT2 responsible for midbody localization, we transfected cells with plasmids containing the NH2-terminal half (ECT2-N), the COOH-terminal half (ECT2-C), or full-length (ECT2-F) ECT2 as GFP fusion proteins. GFP proteins were visualized 72 h after transfection (Fig. 3 A). Like endogenous ECT2, GFP-tagged ECT2-F was detected in the midbody of dividing cells. Although ECT2-N localized in the cytoplasm of interphase cells (data not shown), it accumulated in the midbody of dividing cells. In contrast, ECT2-C was detected in the cytoplasm of mitotic cells. These observations indicate that the NH2-terminal half of ECT2 is required for midbody localization. The nuclear localization of ECT2-F in interphase cells may be attributed to nuclear localization signals located in the central region of ECT2 (Fig. 1 a).


Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis.

Tatsumoto T, Xie X, Blumenthal R, Okamoto I, Miki T - J. Cell Biol. (1999)

Effects of a dominant negative mutant of ECT2 on cytokinesis. (A) Subcellular localization of exogenously expressed ECT2 protein. The full-length (ECT2-F; panels a and b), NH2-terminal half (ECT2-N; panels c–f), or COOH-terminal half (ECT2-C; panels g and h) of ECT2 was transiently expressed in U2OS cells as a GFP-fused protein. GFP fusion proteins were detected by green fluorescence (panels a, c, e, and g). DNA was stained with DAPI (panels b, d, f, and h). (B) The NH2-terminal domain of ECT2 acts as a dominant negative mutant for cytokinesis. Cells were transfected with GFP-fused ECT2-F, ECT2-N, or ECT2-C, or GFP vector alone. GFP-expressed cells are visualized by green fluorescence (panels a and c). DNA was stained with DAPI (panels b and d). Arrowheads indicate multinucleated cells. Bars, 20 μm. (Right panel) GFP-expressing multinucleated cells were scored under immunofluorescent microscopy 72 h after transfection. Data are average of three independent experiments.
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Figure 3: Effects of a dominant negative mutant of ECT2 on cytokinesis. (A) Subcellular localization of exogenously expressed ECT2 protein. The full-length (ECT2-F; panels a and b), NH2-terminal half (ECT2-N; panels c–f), or COOH-terminal half (ECT2-C; panels g and h) of ECT2 was transiently expressed in U2OS cells as a GFP-fused protein. GFP fusion proteins were detected by green fluorescence (panels a, c, e, and g). DNA was stained with DAPI (panels b, d, f, and h). (B) The NH2-terminal domain of ECT2 acts as a dominant negative mutant for cytokinesis. Cells were transfected with GFP-fused ECT2-F, ECT2-N, or ECT2-C, or GFP vector alone. GFP-expressed cells are visualized by green fluorescence (panels a and c). DNA was stained with DAPI (panels b and d). Arrowheads indicate multinucleated cells. Bars, 20 μm. (Right panel) GFP-expressing multinucleated cells were scored under immunofluorescent microscopy 72 h after transfection. Data are average of three independent experiments.
Mentions: Next, we examined the subcellular localization of endogenous ECT2 during cell cycle progression by immunofluorescent analysis of HeLa cells. Interestingly, in interphase cells ECT2 was located predominantly in the nucleus, where no expression of Rho family proteins has been reported (Fig. 2 a). After nuclear membrane breakdown in prometaphase, ECT2 spread into the cytoplasm (Fig. 2 b). During metaphase, ECT2 accumulated in the regions where the mitotic spindle is present (Fig. 2 c). Costaining of ECT2 and tubulin generated the yellow colocalization signal. As cells entered anaphase, ECT2 appeared to concentrate in the central region of the spindle (Fig. 2 d). In late anaphase and telophase, ECT2 was mainly located in the midzone, where the cleavage furrow is formed (Fig. 2 e). During cytokinesis, ECT2 accumulated at the midbody, a region in the middle of the bridge that connects the two daughter cells (Fig. 2 f). When the cells exited mitosis and the nuclear envelope reassembled, ECT2 translocated to the nucleus again (data not shown). To locate the region of ECT2 responsible for midbody localization, we transfected cells with plasmids containing the NH2-terminal half (ECT2-N), the COOH-terminal half (ECT2-C), or full-length (ECT2-F) ECT2 as GFP fusion proteins. GFP proteins were visualized 72 h after transfection (Fig. 3 A). Like endogenous ECT2, GFP-tagged ECT2-F was detected in the midbody of dividing cells. Although ECT2-N localized in the cytoplasm of interphase cells (data not shown), it accumulated in the midbody of dividing cells. In contrast, ECT2-C was detected in the cytoplasm of mitotic cells. These observations indicate that the NH2-terminal half of ECT2 is required for midbody localization. The nuclear localization of ECT2-F in interphase cells may be attributed to nuclear localization signals located in the central region of ECT2 (Fig. 1 a).

Bottom Line: Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis.Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis.These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

View Article: PubMed Central - PubMed

Affiliation: Molecular Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.

ABSTRACT
Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

Show MeSH