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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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Inhibition of cytokinesis by microinjection of anti-ECT2 antibodies. (A) Affinity-purified anti-ECT2 antibodies specific to the NH2-terminal domain (αECT2-N), or the DH domain (αECT2-DH) were microinjected into unsynchronized cultures of HeLa cells. Injected cells were identified by immunostaining with anti–rabbit IgG antibody (panels a, d, and g). Cells were also stained for actin with phalloidine (panels b, e, and h) and for DNA with DAPI (panels c, f, and i) to determine the periphery of the cells and morphology of nuclei, respectively. Panels a–c show the morphology of cells injected with control antibody, where two daughter cells were observed. Microinjection of cells with αECT2-DH (panels d–f) or αΕCT2-N (panels g–i) resulted in single cells with two nuclei 24 h after injection. Longer incubation (48 h) also resulted in cells with more than three nuclei (data not shown). Bars, 10 μm. (B) Number of single interphase cells, normally divided cells, binucleated cells, and multinucleated cells are shown 24 or 48 h after microinjection of the designated antibodies. The last lane includes tri- and tetranucleated cells.
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Figure 4: Inhibition of cytokinesis by microinjection of anti-ECT2 antibodies. (A) Affinity-purified anti-ECT2 antibodies specific to the NH2-terminal domain (αECT2-N), or the DH domain (αECT2-DH) were microinjected into unsynchronized cultures of HeLa cells. Injected cells were identified by immunostaining with anti–rabbit IgG antibody (panels a, d, and g). Cells were also stained for actin with phalloidine (panels b, e, and h) and for DNA with DAPI (panels c, f, and i) to determine the periphery of the cells and morphology of nuclei, respectively. Panels a–c show the morphology of cells injected with control antibody, where two daughter cells were observed. Microinjection of cells with αECT2-DH (panels d–f) or αΕCT2-N (panels g–i) resulted in single cells with two nuclei 24 h after injection. Longer incubation (48 h) also resulted in cells with more than three nuclei (data not shown). Bars, 10 μm. (B) Number of single interphase cells, normally divided cells, binucleated cells, and multinucleated cells are shown 24 or 48 h after microinjection of the designated antibodies. The last lane includes tri- and tetranucleated cells.

Mentions: To further examine the involvement of ECT2 in cytokinesis, we inhibited ECT2 function by microinjection of affinity-purified anti-ECT2 antibodies into asynchronously growing HeLa cells (Fig. 4). Whereas cells injected with control IgG divided normally, ∼60% of cells injected with anti–ECT2-DH, which recognizes the catalytic domain of ECT2, became larger with double nuclei 24 h after injection. Since 25% of the injected cells had not divided at this stage, ∼80% of the cells were multinucleated upon completion of mitosis. After 48 h, 29% of anti–ECT2-DH–injected cells contained three or four nuclei. The presence of cells with more than two nuclei may indicate inhibition of multiple rounds of cytokinesis with anti–ECT2-DH. In contrast, very few control IgG-injected cells were multinucleated. To rule out the possibility that anti-ECT2 cross-reacted with other molecules that regulate cytokinesis, we prepared a second affinity-purified antibody that specifically recognizes the NH2-terminal domain of ECT2. Microinjection of this antibody (anti–ECT2-N) also strongly inhibited cytokinesis (Fig. 3 A, panels g–i; Fig. 3 B). These results strongly suggest that ECT2 plays a critical role in cytokinesis.


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)

Inhibition of cytokinesis by microinjection of anti-ECT2 antibodies. (A) Affinity-purified anti-ECT2 antibodies specific to the NH2-terminal domain (αECT2-N), or the DH domain (αECT2-DH) were microinjected into unsynchronized cultures of HeLa cells. Injected cells were identified by immunostaining with anti–rabbit IgG antibody (panels a, d, and g). Cells were also stained for actin with phalloidine (panels b, e, and h) and for DNA with DAPI (panels c, f, and i) to determine the periphery of the cells and morphology of nuclei, respectively. Panels a–c show the morphology of cells injected with control antibody, where two daughter cells were observed. Microinjection of cells with αECT2-DH (panels d–f) or αΕCT2-N (panels g–i) resulted in single cells with two nuclei 24 h after injection. Longer incubation (48 h) also resulted in cells with more than three nuclei (data not shown). Bars, 10 μm. (B) Number of single interphase cells, normally divided cells, binucleated cells, and multinucleated cells are shown 24 or 48 h after microinjection of the designated antibodies. The last lane includes tri- and tetranucleated cells.
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Related In: Results  -  Collection

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Figure 4: Inhibition of cytokinesis by microinjection of anti-ECT2 antibodies. (A) Affinity-purified anti-ECT2 antibodies specific to the NH2-terminal domain (αECT2-N), or the DH domain (αECT2-DH) were microinjected into unsynchronized cultures of HeLa cells. Injected cells were identified by immunostaining with anti–rabbit IgG antibody (panels a, d, and g). Cells were also stained for actin with phalloidine (panels b, e, and h) and for DNA with DAPI (panels c, f, and i) to determine the periphery of the cells and morphology of nuclei, respectively. Panels a–c show the morphology of cells injected with control antibody, where two daughter cells were observed. Microinjection of cells with αECT2-DH (panels d–f) or αΕCT2-N (panels g–i) resulted in single cells with two nuclei 24 h after injection. Longer incubation (48 h) also resulted in cells with more than three nuclei (data not shown). Bars, 10 μm. (B) Number of single interphase cells, normally divided cells, binucleated cells, and multinucleated cells are shown 24 or 48 h after microinjection of the designated antibodies. The last lane includes tri- and tetranucleated cells.
Mentions: To further examine the involvement of ECT2 in cytokinesis, we inhibited ECT2 function by microinjection of affinity-purified anti-ECT2 antibodies into asynchronously growing HeLa cells (Fig. 4). Whereas cells injected with control IgG divided normally, ∼60% of cells injected with anti–ECT2-DH, which recognizes the catalytic domain of ECT2, became larger with double nuclei 24 h after injection. Since 25% of the injected cells had not divided at this stage, ∼80% of the cells were multinucleated upon completion of mitosis. After 48 h, 29% of anti–ECT2-DH–injected cells contained three or four nuclei. The presence of cells with more than two nuclei may indicate inhibition of multiple rounds of cytokinesis with anti–ECT2-DH. In contrast, very few control IgG-injected cells were multinucleated. To rule out the possibility that anti-ECT2 cross-reacted with other molecules that regulate cytokinesis, we prepared a second affinity-purified antibody that specifically recognizes the NH2-terminal domain of ECT2. Microinjection of this antibody (anti–ECT2-N) also strongly inhibited cytokinesis (Fig. 3 A, panels g–i; Fig. 3 B). These results strongly suggest that ECT2 plays a critical role in cytokinesis.

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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