Imaging trans-cellular neurexin-neuroligin interactions by enzymatic probe ligation.
Imaging trans-cellular neurexin-neuroligin interactions by enzymatic probe ligation.
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DOI：
10.1371/journal.pone.0052823
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Liu DS;Loh KH;Lam SS;White KA;Ting AY
Neurexin和neuroligin是跨膜粘附蛋白，在组织神经元突触间隙中起重要作用。我们的实验室以前报告了一种用于成像neurexin和neuroligin的跨突触结合的方法，称为BLINC（细胞间接触的生物素标记）。在BLINC中，生物素连接酶（BirA）与一种蛋白质融合，而其15个氨基酸的受体肽底物（AP）与结合伴侣融合。当两种融合蛋白在细胞连接处相互作用时，BirA催化AP的位点特异性生物素化，这可以通过用链霉亲和素-荧光团缀合物染色来读出。在这里，我们报告说，BLINC在神经元不能复制使用报告构建体和标记协议先前描述的。我们揭示了缺乏可重复性的技术原因，然后重新设计BLINC报告基因和标记方案，以实现神经元培养中的neurexin-neuroligin BLINC成像。此外，我们介绍了一种新的方法，基于硫辛酸连接酶，而不是生物素连接酶，图像跨细胞neurexin-neuroligin在人胚肾细胞和神经元培养物中的相互作用。这种方法称为ID-PRIME for Interaction-Dependent PRobe Incorporation by Enzymes，由于硫辛酸连接酶融合构建体的更高表面表达，比BLINC更稳健，提供更强和更局部化的标记，并且在信号读出方面比BLINC更通用。ID-PRIME扩展了可用于研究生命系统中跨细胞蛋白质-蛋白质相互作用的方法工具包。
Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.
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DOI：
10.1523/jneurosci.0470-10.2010
发表时间：
2010-06-02
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Siddiqui TJ;Pancaroglu R;Kang Y;Rooyakkers A;Craig AM
通讯作者：
Craig AM
影响因子：
4.8
作者：
Dalby, B;Cates, S;Ciccarone, VC
通讯作者：
Ciccarone, VC
影响因子：
48
作者：
Howarth, M;Chinnapen, DJF;Ting, AY
通讯作者：
Ting, AY
影响因子：
14.8
作者：
Kaech, Stefanie;Banker, Gary
通讯作者：
Banker, Gary
影响因子：
3.7
作者：
Saura CA;Servián-Morilla E;Scholl FG
通讯作者：
Scholl FG