Structural analysis of the molecular organization of the membrane undercoat

细胞膜由不同功能和大小的结构域组成，在生命中起着重要的作用。形成膜底涂层的外周膜蛋白在不同的域内调节着内在膜蛋白的功能和运动。本项目旨在揭示这种膜底涂层的分子结构。为此，采用免疫-冷冻刻蚀复制法结合电镜层析技术对神经生长锥膜底层进行了三维分析。通常，膜底被由肌动蛋白丝、微管、肌动蛋白相关蛋白（Arp2/3）和少量的谱蛋白组成。这些细丝形成复杂的网状结构，紧密地附着在质膜的细胞质表面。神经生长因子在这层膜底层中起什么作用以及如何起作用是非常有趣的。我们特别关注了CRMP-2，因为它参与了轴突的初始形成。虽然CRMP-2主要定位于轴突的微管上，但它也经常被发现存在于生长锥的膜底层中与其他未知颗粒相关的网格蛋白外壳上。这表明CRMP-2可能也参与了生长锥的内吞作用。然而，CRMP-2的确切功能尚不清楚。如果CRMP-2作为轴突rho激酶的底物，通过调节微管和/或肌动蛋白丝的组装来控制轴突的生长，那么它可能在生长锥中有不同的功能，如膜回收。我们也一直在尝试观察在没有任何装饰的生活状态下的膜底涂层。幸运的是，在配备液氦冷却台的电子显微镜下，使用原始方法拆顶的电池直接观察到水合膜底涂层。用类似的方法发现了微管、肌动蛋白丝和网格蛋白包覆的凹坑。这一成功是即将到来的研究项目的重要一步。少

Cell membrane consists of domains with distinct functions and different sizes, which also plays most important roles in life. Peripheral membrane proteins forming membrane undercoat regulate function and movement of intrinsic membrane proteins within various domains. This project aimed to reveal molecular organization of such membrane undercoat. For this purpose immuno-freeze-etching replica method combined with electron microscopic tomography was employed and available for three-dimensional analysis of membrane undercoat of neural growth cone. In common, membrane undercoat consisted of actin filaments, microtubules, actin related proteins (Arp2/3) and small amount of spectrin. Those filaments formed complicated meshwork and attached closely onto the cytoplasmic surface of the plasma membrane. It is very interested what and how neuronal growth factors are associated in this membrane undercoat. In particular, we focused on CRMP-2 because it was involved in initial formation of axon. Alt … More hough CRMP-2 were localized predominantly on microtubules in axons, it was found frequently on clathrin coats being associated with other unknown particles in membrane undercoat of growth cone. This suggests that CRMP-2 is supposed to participate in endocytosis in the growth cone as well.However, the exact function of CRMP-2 has not been unknown. If CRMP-2 controls growing axon by regulating microtubules and/or actin filaments assembly as the substrate of Rho-kinase in axon, it may have different function in growth cone such as the membrane recycling. We also have been trying to observe the membrane undercoat at living state without any decoration.Fortunately, hydrated membrane undercoat was observed directly under the electron microscope equipped with liquid helium cooled stage using the cell unroofed by original method. Microtubles, actin filaments and clathrin coated pits were found in the similar way of freeze-etching method. This success is a very important step for forthcoming research project. Less