Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions
Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions
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DOI：
10.1523/jneurosci.2645-15.2016
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发表时间：
2016-02-17
影响因子：
5.3
通讯作者：
Gomez, Timothy M.
中科院分区：
文献类型：
--
作者：
Nichol, Robert H.;Hagen, Kate M.;Gomez, Timothy M.
生长锥与细胞外基质（ECM）通过整合素受体在粘附位点相互作用，称为点接触。点接触粘附通过许多接头蛋白和信号蛋白将ECM蛋白连接到肌动蛋白细胞骨架。生长锥点接触的一个假定功能是抑制或“抓住”基于肌球蛋白-II的丝状肌动蛋白（F-肌动蛋白）逆行流动（RF），以促进前缘膜突起。在运动的非神经元细胞中，肌球蛋白II结合并对前缘的肌动蛋白丝施加力，在那里发生抓力。然而，在生长锥，目前还不清楚是否类似的F-肌动蛋白抓力影响轴突的生长和指导。在这里，我们表明，在非洲爪蟾脊髓神经元，RF层粘连蛋白（LN）的快速迁移的生长锥相比，非整合素结合的聚-D-赖氨酸（PDL）减少。此外，急性刺激LN加速轴突生长在一个时间过程中，与点接触形成和减少RF。这些结果表明，RF是由点接触，我们表明发生在本地的RF和桩蛋白的双通道成像组件的限制。此外，使用PDL和LN的微图案，我们证明了个体生长锥具有不同的RF速率，同时与两个不同的基质相互作用。在用影响点接触粘连的化学吸引和化学排斥轴突引导线索处理的生长锥中也观察到对RF率的相反影响。最后，我们表明，RF在体内显着衰减，这表明它是脊髓内的分子离合力的限制。总之，我们的研究结果表明，RF的局部抓持可以控制轴突引导线索下游ECM蛋白的轴突引导。
Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-D-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues.