Stable, Non-Destructive Immobilization of Native Nuclear Membranes to Micro-Structured PDMS for Single-Molecule Force Spectroscopy
Stable, Non-Destructive Immobilization of Native Nuclear Membranes to Micro-Structured PDMS for Single-Molecule Force Spectroscopy
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DOI：
10.1002/cphc.200900219
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发表时间：
2009-07-13
期刊：
影响因子：
2.9
通讯作者：
Hinterdorfer, Peter
中科院分区：
文献类型：
--
作者：
Rangl, Martina;Nevo, Reinat;Hinterdorfer, Peter
在真核细胞中，细胞核与细胞质被双层核膜（NE）隔开。两个隔室之间的分子交换由嵌入NE膜中的核孔复合物（NPC）介导。蛋白质和RNA等分子通过核膜的转运由转运穿梭因子（核转运蛋白）执行。因此，它们对接到位于NPC各处的特定结合位点，即所谓的苯丙氨酸-甘氨酸核孔蛋白（FG Nups）。分子识别力光谱（MRFS）允许在单分子水平上研究结合。因此，AFM针尖携带配体，例如，特定的核转运蛋白，而核膜及其受体安装在表面上。因此，使用MRFS研究核质转运机制的首要要求之一是开发一种优化的膜制剂，以保留NPC的结构和功能。本研究提出了一种稳定的非洲爪蟾核膜的非破坏性制备方法。我们使用微结构的聚二甲基硅氧烷（PDMS），由于其弹性，化学和机械性能，为固定和生物完整性提供了理想的平台。这是一个坚实的基础，研究分子识别，运输相互作用，并通过NPC易位过程。作为第一个识别系统，我们研究了一个重要的运输穿梭因子，importin β，和它的结合位点的NPC，FG-域之间的相互作用。
In eukaryotic cells the nucleus is separated from the cytoplasm by a double-membraned nuclear envelope (NE). Exchange of molecules between the two compartments is mediated by nuclear pore complexes (NPCs) that are embedded in the NE membranes. The translocation of molecules such as proteins and RNAs through the nuclear membrane is executed by transport shuttling factors (karyopherines). They thereby dock to particular binding sites located all over the NPC, the so-called phenylalanine-glycin nucleoporines (FG Nups). Molecular recognition force spectroscopy (MRFS) allows investigations of the binding at the single-molecule level. Therefore the AFM tip carries a ligand for example, a particular karyopherin whereas the nuclear membrane with its receptors is mounted on a surface. Hence, one of the first requirements to study the nucleocytoplasmatic transport mechanism using MRFS is the development of an optimized membrane preparation that preserves structure and function of the NPCs. In this study we present a stable non-destructive preparation method of Xenopus laevis nuclear envelopes. We use micro-structured polydimethylsiloxane (PDMS) that provides an ideal platform for immobilization and biological integrity due to its elastic, chemical and mechanical properties. It is a solid basis for studying molecular recognition, transport interactions, and translocation processes through the NPC. As a first recognition system we investigate the interaction between an important transport shuttling factor, importin beta, and its binding sites on the NPC, the FG-domains.