Axon Initiation and Growth Cone Turning on Bound Protein Gradients
Axon Initiation and Growth Cone Turning on Bound Protein Gradients
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DOI：
10.1523/jneurosci.1121-09.2009
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发表时间：
2009-06-10
影响因子：
5.3
通讯作者：
Poo, Mu-ming
中科院分区：
文献类型：
--
作者：
Mai, Junyu;Fok, Lee;Poo, Mu-ming
已知分泌的引导因子的细胞外梯度引导轴突寻路和神经元迁移。这些因子可能与细胞表面或细胞外基质结合，但它们是否以及如何在结合梯度中起作用仍不清楚。在这项研究中，我们已经开发出一种新的技术，用于快速生产稳定的微观梯度的基板结合蛋白质的共价键合的蛋白质与环氧涂层的玻璃基板，而他们在琼脂糖凝胶中扩散。利用这种方法，我们发现netrin-1和脑源性神经营养因子（BDNF）的结合梯度可以抑制培养的海马神经元轴突的起始和转向。此外，绑定BDNF梯度引起吸引和排斥的极化反应的梯度低和高的平均密度的BDNF，分别。这种对BDNF的双向反应依赖于神经元中cAMP的基础水平。最后，我们的数据表明，神经元对结合BDNF梯度的吸引力反应取决于神经元中BDNF密度的绝对差异，而不是相对差异，在基底表面上的最小有效差异为1-2 BDNF分子/μ m（2）。因此，底物结合的指导因素是非常有效的极化轴突的起始和生长，和扩散打印技术是有用的研究结合蛋白梯度诱导的神经元反应。
Extracellular gradients of secreted guidance factors are known to guide axon pathfinding and neuronal migration. These factors are likely to bind to cell surfaces or extracellular matrix, but whether and how they may act in bound gradients remains mostly unclear. In this study, we have developed a new technique for rapid production of stable microscopic gradients of substrate-bound proteins by covalent bonding of the proteins with an epoxy-coated glass substrate while they are diffusing in an agarose gel. Using this method, we found that bound gradients of netrin-1 and brain-derived neurotrophic factor (BDNF) can polarize the initiation and turning of axons in cultured hippocampal neurons. Furthermore, bound BDNF gradient caused attractive and repulsive polarizing response on gradients of low- and high-average density of BDNF, respectively. This novel bidirectional response to BDNF depended on the basal level of cAMP in the neuron. Finally, our data showed that the neuron's attractive response to bound BDNF gradient depended on the absolute difference rather than the relative difference in the BDNF density across the neuron, with a minimal effective difference of 1-2 BDNF molecule/mu m(2) on the substrate surface. Thus, substrate-bound guidance factors are highly effective in polarizing axon initiation and growth, and the diffusive printing technique is useful for studying neuronal responses induced by bound protein gradients.