Suter Daniel M
Department of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2011;769:65-86. doi: 10.1007/978-1-61779-207-6_6.
The neuronal growth cone, a highly motile structure at the tip of neuronal processes, is an excellent model system for studying directional cell movements. While biochemical and genetic approaches unveiled molecular interactions between ligand, receptor, signaling, and cytoskeleton-associated proteins controlling axonal growth and guidance, in vitro live cell imaging has emerged as a crucial approach for dissecting cellular mechanisms of growth cone motility and guidance. Important insights into these mechanisms have been gained from studies using the large growth cones elaborated by Aplysia californica neurons, an outstanding model system for live cell imaging for a number of reasons. Identified neurons can be isolated and imaged at room temperature. Aplysia growth cones are five to ten times larger than growth cones from other species, making them suitable for quantitative high-resolution imaging of cytoskeletal protein dynamics and biophysical approaches. Lastly, protein, RNA, fluorescent probes, and small molecules can be microinjected into the neuronal cell body for localization and functional studies. This chapter describes culturing of Aplysia bag cell neurons, live cell imaging of neuronal growth cones using differential interference contrast and fluorescent speckle microscopy as well as the restrained bead interaction assay to induce adhesion-mediated growth cone guidance in vitro.
神经元生长锥是神经元突起末端高度可移动的结构，是研究细胞定向运动的优秀模型系统。虽然生化和遗传学方法揭示了控制轴突生长和导向的配体、受体、信号传导和细胞骨架相关蛋白之间的分子相互作用，但体外活细胞成像已成为剖析生长锥运动性和导向细胞机制的关键方法。利用加州海兔神经元形成的大型生长锥进行的研究，为这些机制提供了重要见解，由于多种原因，加州海兔是活细胞成像的优秀模型系统。可分离出已识别的神经元并在室温下成像。海兔的生长锥比其他物种的生长锥大五到十倍，使其适用于细胞骨架蛋白动力学的定量高分辨率成像和生物物理方法。最后，可以将蛋白质、RNA、荧光探针和小分子显微注射到神经元细胞体中进行定位和功能研究。本章介绍了海兔袋状细胞神经元的培养、使用微分干涉对比和荧光斑点显微镜对神经元生长锥进行活细胞成像，以及在体外诱导粘附介导的生长锥导向的受限珠相互作用试验。
