Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.
Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.
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DOI：
10.3389/fnmol.2013.00026
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发表时间：
2013
影响因子：
4.8
通讯作者：
Garthwaite J
中科院分区：
文献类型：
--
作者：
Bhargava Y;Hampden-Smith K;Chachlaki K;Wood KC;Vernon J;Allerston CK;Batchelor AM;Garthwaite J
基因编码生物传感器是理解细胞信号转导机制的有力工具。为了利用基于egfp的荧光生物传感器FlincG （cGMP荧光指示剂）研究神经元中的cGMP信号传导，我们在尝试用质粒DNA转染后发现，即使在HEK293T细胞中，荧光也很弱或不存在。然而，先前证明FlincG对HEK293T细胞的腺病毒感染是成功的。这两种结构体在EGFP结构域中都发现了突变，并且在c端有17个氨基酸的尾部，与已发表的序列不同。在稳定表达一氧化氮（NO）激活的guanyyl环化酶和磷酸二酯酶-5的HEK293T细胞系中，系统地检查了这些差异，以及发现有利于Ca2+生物传感器相关GCaMP家族的突变。通过8-溴-cGMP或NO的灌注评估，恢复突变氨基酸改善了基础荧光，而额外恢复正确的c端尾部导致cGMP感知能力差。最终鉴定出两个改进的FlincGs：一个（FlincG2）尾部发散，具有中等的基础荧光和cGMP响应幅度；另一个（FlincG3）尾部正确，EGFP区域具有gcamp样突变和n端标签，在这两个方面都更优越。与其他基于egfp的生物传感器一样，所有测试的变体都受到生理范围内pH值的强烈影响。纯化后的FlincG3蛋白的cGMP亲和力（0.89 μM）低于原FlincG蛋白（0.17 μM），但保持了快速动力学和230倍的cAMP选择性。FlincG2或FlincG3在分化的N1E-115神经母细胞瘤细胞以及海马和背根神经节细胞的原代培养中成功表达，使其能够实时成像神经（和其他）细胞及其亚细胞特化中的cGMP动力学。
Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations.
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影响因子：
9.5
作者：
Miller CL;Cai Y;Oikawa M;Thomas T;Dostmann WR;Zaccolo M;Fujiwara K;Yan C
通讯作者：
Yan C
影响因子：
16.6
作者：
Mehta S;Zhang J
通讯作者：
Zhang J
影响因子：
4.4
作者：
Oh, J;Karlmark, K;Lubec, G
通讯作者：
Lubec, G
影响因子：
3.3
作者：
Kummer, AD;Wiehler, J;Michel-Beyerle, ME
通讯作者：
Michel-Beyerle, ME
活性依赖性可塑性在细胞一氧化氮CGMP信号传导中的机制。
DOI：
10.1074/jbc.m109.030338
发表时间：
2009-09-18
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Halvey EJ;Vernon J;Roy B;Garthwaite J
通讯作者：
Garthwaite J