Kerstein Patrick C, Patel Kevin M, Gomez Timothy M
Department of Neuroscience and.
Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53705.
J Neurosci. 2017 Feb 8;37(6):1568-1580. doi: 10.1523/JNEUROSCI.2769-16.2016. Epub 2017 Jan 9.
Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca arise from the precise localization of Ca signals into microdomains containing specific Ca effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in spinal cord neurons both and Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca within filopodia. In addition, blocking calpain cleavage of talin and FAK promotes Rohon-Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK. The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may identify areas of therapeutic intervention in disease or injury. One important signal that controls growth cones is that of local Ca transients, which control the rate and direction of axon outgrowth. We demonstrate here that Ca-dependent inhibition axon outgrowth and guidance is mediated by calpain proteolysis of the adhesion proteins talin and focal adhesion kinase. Our findings provide mechanistic insight into Ca/calpain regulation of growth cone motility and axon guidance during neuronal development.
轴突导向其合适的突触靶位点需要在生长锥内对生化信号进行空间和时间上精确的调节。离子钙（Ca）是轴突导向的一种重要信号，它介导对生长锥运动性的相反作用。Ca的多种效应源于Ca信号精确地定位于含有特定Ca效应器的微区。例如，下层基质机械和化学成分的差异会在生长锥丝状伪足内引发局部Ca信号，通过激活蛋白酶钙蛋白酶来调节轴突导向。然而，钙蛋白酶如何调节生长锥运动性仍不清楚。在这里，我们确定黏附蛋白踝蛋白和黏着斑激酶（FAK）是脊髓神经元中钙蛋白酶的蛋白水解靶标。抑制钙蛋白酶会增加内源性黏附信号向生长锥丝状伪足的定位。使用活细胞显微镜以及踝蛋白（L432G）和FAK（V744G）的特定抗钙蛋白酶点突变体，我们发现钙蛋白酶通过切割踝蛋白和FAK来抑制基于桩蛋白的黏附组装，并通过切割FAK来促进黏附解体。阻断钙蛋白酶对踝蛋白和FAK的切割可抑制丝状伪足内Ca局部释放引起的排斥性转向。此外，阻断钙蛋白酶对踝蛋白和FAK的切割可促进罗霍恩 - 贝德外周轴突向皮肤延伸。这些数据表明，丝状伪足Ca信号通过钙蛋白酶对踝蛋白和FAK的特异性切割来调节黏附动力学，从而调控轴突生长和导向。神经元网络的正确形成需要在发育过程中通过称为生长锥的运动结构对轴突和树突进行精确导向。了解控制生长锥运动性的细胞内信号机制将阐明神经系统如何发育和再生，并可能确定疾病或损伤中的治疗干预领域。控制生长锥的一个重要信号是局部Ca瞬变信号，它控制轴突生长的速率和方向。我们在此证明，Ca依赖性抑制轴突生长和导向是由钙蛋白酶对黏附蛋白踝蛋白和黏着斑激酶的蛋白水解介导的。我们的研究结果为神经元发育过程中Ca/钙蛋白酶对生长锥运动性和轴突导向的调节提供了机制性见解。
