Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.
Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.
复制标题
DOI：
10.1242/jcs.260802
复制
发表时间：
2023-05-15
影响因子：
4
通讯作者：
Goedhart, Joachim
中科院分区：
文献类型：
--
作者：
Mahlandt, Eike K.;Kreider-Letterman, Gabriel;Chertkova, Anna O.;Garcia-Mata, Rafael;Goedhart, Joachim
Rac（在本文中指Rac家族）和Cdc42是Rho GTP酶，其调节板足和丝状伪足的形成，并且因此在诸如细胞迁移的过程中至关重要。Rac和Cdc42的基于双阳离子的生物传感器在其特异性或亲和力方面尚未得到很好的表征。在这项研究中，我们确定搬迁传感器的候选人为Rac和Cdc42。我们比较了它们（1）结合组成型活性Rho GTP酶的能力，（2）对Rac和Cdc42的特异性，以及（3）在基于细胞的测定中的重新定位效率。随后，通过多域方法提高了重定位效率。对于Rac1，我们发现了一个传感器候选低搬迁效率。对于Cdc42，我们发现了几个传感器具有足够的重新定位效率和特异性。这些优化的传感器使得Rho GTdR重定位传感器能够得到更广泛的应用，这通过检测组装侵入伪足时的局部内源性Cdc42活性来展示。此外，我们测试了几种荧光蛋白和HaloTag对Rho定位传感器的募集效率的影响，以找到多重实验的最佳条件。这种定位传感器的表征和优化将扩大其应用和接受范围。 一个系统的，基于细胞的比较G蛋白结合域的潜力，定位为基础的，遗传编码的生物传感器成像Cdc42或Rac活性在单细胞中。
Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance. A systematic, cell-based comparison of G-protein-binding domains for their potential as localization-based, genetically encoded biosensors for imaging of Cdc42 or Rac activity in single cells.
登录
查看更多内容
DOI：
10.1083/jcb.200806047
发表时间：
2009-01-26
期刊：
The Journal of cell biology
影响因子：
--
作者：
Dewitt S;Darley RL;Hallett MB
通讯作者：
Hallett MB
影响因子：
7.7
作者：
Gillingham，Alison K.;Bertram，Jessie;Munro，Sean
通讯作者：
Munro，Sean
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
5.3
作者：
Itoh, RE;Kurokawa, K;Matsuda, M
通讯作者：
Matsuda, M
影响因子：
7.7
作者：
Laan, Liedewij;Koschwanez, John H.;Murray, Andrew W.
通讯作者：
Murray, Andrew W.