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的新型显微镜，我们能够清楚地观察到绿色荧光蛋白标记的输入素β、载体蛋白和绿色荧光蛋白标记的货运蛋白在核膜上运输过程中的单个荧光分子，无论是在渗透性细胞中还是在活细胞中。对单个核孔的图像分析表明，两点分辨率达到了70 nm。通过定量分析，得到了转位分子与核祖细胞相互作用的动力学参数。发现了两种类型的结合：较弱的结合部位，聚集高达~100个分子/NPC，局部集中底物；较强的结合部位，高达8个分子/NPC的结合。单分子分析的保留时间和单核分析的易位率与~8分子/NPC的系数有显著的相关性，这表明了运输的化学计量学。我们将较弱的结合部位称为“多重结合部位”，它很可能由含有核孔蛋白的FG-重复组成。根据相关系数，我们提出了核孔复合体中存在8个“过渡中心”。这个转运点是否等同于观察到的强结合位点，还有待进一步研究阐明。这些发现是否解释了不同的路径，显示了不同的传输速率，也有待阐明。在平行实验中，我们纯化并鉴定了70 kDa热休克同源蛋白(Hsc70)，它是一个促进Importinβ循环的分子。观察到hsc70不仅对Importinβ的核输出有影响，而且对另一种输入受体Transportin和Importinα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而广泛地调节核质转运系统(Kose等人，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).