Single-molecule imaging of NGF axonal transport in microfluidic devices.
Single-molecule imaging of NGF axonal transport in microfluidic devices.
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DOI：
10.1039/c003385e
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发表时间：
2010-10-07
期刊：
影响因子：
6.1
通讯作者：
Cui B
中科院分区：
文献类型：
--
作者：
Zhang K;Osakada Y;Vrljic M;Chen L;Mudrakola HV;Cui B
神经生长因子（NGF）信号传导始于神经末端，在那里它结合并激活膜受体，随后通过轴突将细胞生存信号传递到细胞体。最近的一项研究表明，大多数内体含有单个NGF分子，这使得单分子成像成为NGF研究的重要工具。尽管是一种越来越流行的技术，但活细胞中的单分子成像通常受到背景荧光的限制。在这里，我们采用微流体培养平台来实现活神经元中单分子成像的背景减少。微流体装置引导神经元的生长，并允许单独控制细胞体或轴突末端的微环境。优化了微流控装置的设计，当量子点标记的NGF（Qdot-NGF）仅应用于远端轴突室而成像仅在细胞体室中进行时，三室装置成功地实现了对单个NGF的轴突运输的直接观察。Qdot-NGF被证明只向细胞体移动，具有特征性的走走停停的运动模式。在不同温度下的测量表明，在36-14°C的范围内，NGF逆行转运的速率呈指数下降。温度降低10°C导致NGF逆行转运速率降低三倍。我们对NGF运输的成功测量表明，微流体装置可以作为神经元中分子过程的单分子成像的独特平台。
Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately-controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14°C. A 10°C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single molecule imaging of molecular processes in neurons.
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影响因子：
6.1
作者：
Millet, Larry J.;Stewart, Matthew E.;Gillette, Martha U.
通讯作者：
Gillette, Martha U.
DOI：
10.1073/pnas.0706192104
发表时间：
2007-08-21
影响因子：
11.1
作者：
Cui, Bianxiao;Wu, Chengbiao;Chu, Steven
通讯作者：
Chu, Steven
DOI：
10.1073/pnas.0811484106
发表时间：
2009-04-14
影响因子：
11.1
作者：
McWhirter, J. Liam;Noguchi, Hiroshi;Gompper, Gerhard
通讯作者：
Gompper, Gerhard
DOI：
10.1002/neu.480060112
发表时间：
1975-01-01
期刊：
JOURNAL OF NEUROBIOLOGY
影响因子：
--
作者：
OCHS, S;SMITH, C
通讯作者：
SMITH, C
DOI：
10.1006/bbrc.2000.2856
发表时间：
2000-06-16
影响因子：
3.1
作者：
Kawaguchi, K;Ishiwata, S
通讯作者：
Ishiwata, S