Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy
Permeability of the nuclear envelope at isolated Xenopus oocyte nuclei studied by scanning electrochemical microscopy
复制标题
DOI：
10.1021/ac048370j
复制
发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Amemiya, S
中科院分区：
文献类型：
--
作者：
Guo, JD;Amemiya, S
在间期真核细胞中，细胞质和细胞核之间的分子运输是由核孔复合体（NPC）介导的，该复合体穿过双膜核膜（NE）。通过扫描电化学显微镜 (SECM) 研究了从非洲爪蟾卵母细胞分离的大的完整细胞核（直径大约为 400 μm）的 NE 的局部渗透性。在含有氧化还原活性分子的等渗缓冲溶液中，使用直径为 10 微米和 2 微米的 Pt 盘微电极在细胞核处测量稳态尖端电流与尖端-核距离曲线（接近曲线）。标准化形式的逼近曲线与尖端直径无关，表明氧化还原分子的扩散限制膜传输。 SECM 计时电流分析法证明，尖端-核距离较短时稳态尖端电流的降低是由于核中氧化还原分子的扩散系数和浓度比缓冲溶液中的更小。实验方法曲线与自由渗透膜的理论曲线非常吻合，产生的分子 NE 渗透性比双层脂膜和细胞膜的渗透性至少大 2 个数量级。这一结果表明，开放的 NPC 孔促进了氧化还原分子穿过 NE 的被动运输。通过假设 NE 为纳米尺寸的 NPC 孔阵列，根据 SECM 数据估计单个 NPC 通道可持续的氧化还原分子通量（每秒每个 NPC > 9.8 x 106 个分子）和通道孔的直径（> 15 nm）。通过荧光显微镜研究有或没有核定位信号的罗丹明标记牛血清白蛋白的信号介导的核输入，检查氧化还原分子对细胞核和NPC功能的影响。
In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (similar to 400 mu m in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-mu m-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (> 9.8 x 106 molecules per NPC per second) and the diameter of the channel pore (> 15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.