Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.
Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.
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DOI：
10.4049/jimmunol.1501655
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发表时间：
2016-04-15
期刊：
影响因子：
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通讯作者：
Hodgson L
中科院分区：
文献类型：
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作者：
Miskolci V;Wu B;Moshfegh Y;Cox D;Hodgson L
尽管造血细胞特异性Rac 2与典型同种型Rac 1具有92%的同源性，但这些同种型已显示在免疫细胞中发挥非冗余作用。为了研究Rac在活细胞中的异构体特异性动力学，我们开发了Rac 2的遗传编码的基于单链FRET的生物传感器。我们还对现有的单链Rac 1生物传感器进行了重大改进。我们优化了生物传感器构建体，使其在造血细胞中易于表达，并在RAW264.7细胞的小鼠巨噬细胞亚系中进行了功能验证。Rac2，沿着与Rac1和Cdc42，已被牵连在形成肌动蛋白丰富的突起的巨噬细胞，但他们的个人激活动力学还没有以前的特点。我们发现Rac1和Rac2具有相似的激活动力学，但它们对外源刺激fMLP的响应具有非常不同的空间分布。活性Rac 1主要定位于细胞周边，而活性Rac 2分布在整个细胞中，在核周区域的浓度明显较高。我们还进行了广泛的形态动力学分析的Rac1，Rac2和Cdc42活动在随机突起的延伸。我们发现Rac2似乎在随机突起的产生中起主导作用，因为我们观察到Rac2在远离前缘的区域中的初始强烈激活，随后是Rac1的激活，Rac2的第二次爆发，然后是紧接前缘后面的Cdc42。总的来说，异构体特异性生物传感器，已经优化的表达应该是有价值的询问在活细胞中的Rho家族GTdR活性的协调。
Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play non-redundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells we developed a genetically-encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, along with Rac1and Cdc42, have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics yet they had very distinct spatial distributions in response to the exogenous stimulus, fMLP. Active Rac1 was mainly localized to the cell periphery, while active Rac2 was distributed throughout the cell with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2 and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.