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基于细胞的优化和遗传编码位置传感器对 Cdc42 或 Rac 活性的表征。

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

机构信息

Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA.

出版信息

J Cell Sci. 2023 May 15;136(10). doi: 10.1242/jcs.260802. Epub 2023 May 25.

DOI:10.1242/jcs.260802
PMID:37226883
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10234108/
Abstract

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.

摘要

Rac（此处指 Rac 家族）和 Cdc42 是 Rho GTPases，它们调节片状伪足和丝状伪足的形成，因此在细胞迁移等过程中至关重要。基于重定位的 Rac 和 Cdc42 生物传感器在其特异性或亲和力方面尚未得到很好的描述。在这项研究中，我们确定了 Rac 和 Cdc42 的重定位传感器候选物。我们比较了它们的（1）结合组成性激活的 Rho GTPases 的能力，（2）对 Rac 和 Cdc42 的特异性，以及（3）在细胞测定中的重定位效率。随后，通过多结构域方法提高了重定位效率。对于 Rac1，我们找到了一个重定位效率较低的传感器候选物。对于 Cdc42，我们找到了几个具有足够重定位效率和特异性的传感器。这些优化后的传感器使 Rho GTPase 重定位传感器的应用范围更广，通过检测组装入侵伪足处的局部内源性 Cdc42 活性来展示。此外，我们测试了几种荧光蛋白和 HaloTag 对 Rho 定位传感器募集效率的影响，以找到用于多重实验的最佳条件。对重定位传感器的这种表征和优化将扩大它们的应用和接受程度。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3fd2/10234108/c9ebe4922a22/joces-136-260802-g7.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3fd2/10234108/789611a6c0ad/joces-136-260802-g6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3fd2/10234108/c9ebe4922a22/joces-136-260802-g7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3fd2/10234108/0d778695c0a8/joces-136-260802-g1.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3fd2/10234108/c9ebe4922a22/joces-136-260802-g7.jpg

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