The ect2 rho Guanine nucleotide exchange factor is essential for early mouse development and normal cell cytokinesis and migration.
The ect2 rho Guanine nucleotide exchange factor is essential for early mouse development and normal cell cytokinesis and migration.
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DOI：
10.1177/1947601912437035
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发表时间：
2011-10-01
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影响因子：
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通讯作者：
Der, Channing J
中科院分区：
文献类型：
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作者：
Cook, Danielle R;Solski, Patricia A;Der, Channing J
Ect 2是鸟嘌呤核苷酸交换因子（RhoGEF）的人Dbl家族的成员，其充当Rho家族小GTP酶的激活剂。虽然Ect 2是至少25种可以激活RhoA小GT3的RhoGEF之一，但使用已建立的细胞系进行的细胞培养研究确定Ect 2对哺乳动物细胞胞质分裂和增殖至关重要。为了解决Ect 2在正常哺乳动物发育中的功能，我们进行基因打靶以产生Ect 2敲除小鼠。杂合Ect 2（+/-）小鼠表现出正常的发育和寿命，表明Ect 2单倍缺陷对发育或生长无害。相反，Ect 2（-/-）胚胎在出生或植入后阶段未发现。Ect 2（-/-）胚泡在胚胎第3.5天回收，但在培养物中没有产生活的生长物，表明Ect 2是围着床期发育所需的。为了进一步评估Ect 2在正常细胞生理学中的重要性，我们从Ect 2（fl/fl）胚胎（MEF）中分离原代成纤维细胞，并使用Cre重组酶的腺病毒递送来消融Ect 2。我们观察到多核细胞的显著增加和G2/M期细胞的积累，与Ect 2在胞质分裂中的作用一致。Ect 2缺陷也引起细胞质的扩大和受损的细胞迁移。最后，虽然Ect 2依赖的RhoA激活与胞质分裂有关，但Ect 2也可以激活Rac 1和Cdc 42，从而引起生长转化。令人惊讶的是，异位表达的组成型激活的RhoA，Rac 1，或Cdc 42，已知的底物的Ect 2，未能表型Ect 2，并没有拯救的缺陷，在胞质分裂所造成的损失Ect 2。总之，我们的研究结果确立了Ect 2在发育和正常细胞增殖中的独特作用。
Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2(+/-) mice showed normal development and life span, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2(-/-) embryos were not found at birth or postimplantation stages. Ect2(-/-) blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2(fl/fl) embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis caused by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.