Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses.
Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses.
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DOI：
10.1039/c7nr09130c
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发表时间：
2018-05-31
期刊：
影响因子：
6.7
通讯作者：
Kittler JT
中科院分区：
文献类型：
--
作者：
Modi S ;Higgs NF ;Sheehan D ;Griffin LD ;Kittler JT
应用抗GFP纳米抗体缀合的QD光学探针来研究体外和离体的单颗粒追踪。这种小的、高度特异性的探针识别在细胞膜上表达的GFP，并报告潜在靶蛋白的扩散动力学。神经元通过突触相互交流，突触显示出对专门受体的富集。尽管许多研究已经使用单粒子追踪探索了这些受体在解离神经元中的空间富集和扩散，但对它们在复杂组织（如脑切片）中突触的动态特性知之甚少。在这里，我们报告了使用更小的和高度特异性的量子点与重组单域抗体片段（VHH片段）对绿色荧光蛋白的结合，以提供信息的扩散粘附分子在生长锥和神经递质受体在突触。我们的数据表明，QD-纳米抗体可以测量在原代神经元培养物以及离体大鼠脑切片中兴奋性和抑制性突触的神经递质受体动力学。我们还证明了这种方法可以应用于标记多个蛋白质，同时监测它们的行为。因此，我们提供了一种策略，标记的膜蛋白的多重成像研究它们的聚类，扩散和运输在体外以及在天然组织环境，如脑切片。
An anti-GFP nanobody conjugated QD optical probe was applied to study single particle tracking in vitro and ex vivo. This small, highly specific probe recognized GFP when expressed at the cell membrane and reported diffusion dynamics of the underlying target protein. Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for multiplex imaging of tagged membrane proteins to study their clustering, diffusion and transport both in vitro as well as in native tissue environments such as brain slices.
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影响因子：
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