Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.
Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.
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用基于FRET的探针可视化的细胞分裂过程中Rho-Family GTPase的活性。
DOI：
10.1083/jcb.200212049
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发表时间：
2003-07-21
影响因子：
7.8
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
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作者：
Yoshizaki, Hisayoshi;Ohba, Yusuke;Kurokawa, Kazuo;Itoh, Reina E;Nakamura, Takeshi;Mochizuki, Naoki;Nagashima, Kazuo;Matsuda, Michiyuki
Rho家族GTP酶调节许多细胞功能。为了使活细胞中Rho家族GTP酶的活性可视化，我们先前开发了用于Rac 1和Cdc 42的基于荧光共振能量转移（FRET）的探针（Itoh，R.E.，K. Kurokawa，Y. Ohba，H. Yoshizaki，N. Mochizuki和M.松田2002.摩尔Cell. 22：6582-6591）。在这里，我们为RhoA添加了两种类型的探针。一个是监测鸟嘌呤核苷酸交换因子和GTP酶激活蛋白之间的活性平衡，另一个是监测GTP-RhoA的水平。使用这些FRET探针，我们成像的活性Rho家族的GTP酶在HeLa细胞的细胞分裂过程中。RhoA、Rac 1和Cdc 42的活性在间期细胞质膜上较高，进入M期后迅速下降。从后期开始，RhoA活性在质膜上（包括卵裂沟）逐渐升高。Rac 1活性在纺锤体中间区被抑制，在极侧的质膜在末期后增加。Cdc 42活性在质膜上受到抑制，在胞质分裂过程中在细胞内膜上的活性很高。总之，我们可以使用基于FRET的探针来可视化细胞分裂期间Rho家族GTP酶的复杂时空调控。
Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)–based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582–6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.