Analysis of nuclear pore translocation mechanism : single molecule analysis and biochemical approach

利用基于全内反射荧光显微镜（TIRF）的新型显微镜，我们能够清楚地看到绿色荧光蛋白（GFP）标记的输入蛋白β、载体蛋白和GFP标记的货物蛋白在渗透细胞和活细胞的核膜上运输过程中的单个荧光分子。对单个核孔的图像分析表明，该方法达到了70 nm的两点分辨率。通过定量分析获得了易位分子与npc相互作用的动力学参数。发现了两种类型的绑定；较弱的结合位点，聚集约100个分子/NPC，局部集中底物；更强的结合位点，每个NPC可以结合8个分子。单分子分析的保留时间和单核分析的易位率在~ 8个分子/NPC的系数上呈显著相关，表明了转运的化学计量性质。我们将较弱的结合位点称为“多重位点”，这可能是由含有核孔蛋白的fg重复组成的。根据相关系数，我们推测核孔复合物中存在8个“转运位点”。该转运位点是否与观察到的强结合位点等同，有待进一步研究阐明。这些发现是否能解释不同的途径，这表明了广泛的运输速率，也有待阐明。在平行实验中，我们纯化并鉴定了70kDa热休克同源蛋白（hsc70）作为促进进口蛋白β循环的分子。hsc70不仅对进口蛋白β的核输出有影响，而且对另一种进口受体转运蛋白和进口蛋白α的核输出也有影响。这些结果表明hsc70通过调节受体蛋白的核输出广泛调节核细胞质运输系统（Kose等，J Cell Biol. 2005）。

Using novel microscopy based on total internal reflection fluorescent microscopy (TIRF), we were able to clearly visualize single fluorescent molecules of green fluorescent protein (GFP)-tagged importin β, a carrier protein, and GFP-tagged cargo protein during transport on the nuclear envelope in both permeabilized cells and in living cells. Image analysis of single nuclear pores showed that two point resolution of 70 nm was achieved. Kinetic parameters of the interactions between translocating molecules and NPCs were obtained through quantitative analysis. Two types of binding were found ; weaker binding sites, which gathers up to 〜100 molecules/NPC, concentrating substrates locally ; and stronger binding sites, where up to 〜8 molecules/NPC are bound. Retention times by single molecule analysis and translocation rates by single nuclear analysis showed a significant correlation with a coefficient of 〜8 molecules/NPC, which exhibits the stoichiometry of transport. We termed weaker binding sites as a "multiplex sites", which is likely to consist from FG-repeat containing nucleoporins. Based on correlation coefficient, we proposed presence of 8 "transit site" in nuclear pore complex. Whether this transit site is equivalent to strong binding sites observed remains to be elucidated in the further study. Whether the findings account for different pathways, which show a wide range of transport rates, also remains to be elucidated. In the parallel experiments, we have purified, and identified 70kDa heat-shock cognate protein (hsc70) as a molecule that facilitates recycling of importin β. The effect of hsc70 was observed, not only in the case of the nuclear export of importin β but also for that of another import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating nuclear export of receptor proteins (Kose et al., J Cell Biol. 2005).