Single Channel Recording of the Nuclear Pore Complex

描述(申请人提供)：细胞核是真核细胞的中央细胞器。细胞核和细胞质之间的分子运输只通过核孔复合体(NPC)来调节，核孔复合体穿透定义核室的双膜核膜。我们的长期目标是获得单个鼻咽癌个体通道门控和蛋白质易位事件的实时信息。有关通过纳米蛋白质机器进行核质运输的信息将有助于我们全面了解许多生物学过程和疾病，并将加速未来药物对基因表达的调控。为了实现这一目标，我们将通过将扫描电化学显微镜(SECM)的空间和时间分辨率提高到纳米和微秒范围，建立一种新的单通道鼻咽癌记录实验方法。虽然SECM是一种成熟的非侵入性和定量的膜通透性测量方法，但通过纳米级SECM探针提高空间分辨率将使我们能够测量单个鼻咽癌的通透性。通过监测单个通道事件对单个NPC渗透率的影响，可以获得关于单个通道事件的高时间分辨率的实时信息。时间分辨信息将使我们能够评估目前无法用现有实验数据检验其有效性的输运模型，从而更全面地理解这一重要的输运过程。

DESCRIPTION (provided by applicant): The nucleus is the central organelle of eukaryotic cells. The molecular transport between the nucleus and the cytoplasm is mediated only through the nuclear pore complex (NPC), which perforates the double membraned nuclear envelope that defines the nuclear compartment. Our long-term objective is to obtain real-time information about individual channel-gating and protein-translocation events in the single NPC. The information about the nucleocytoplasmic transport through the nanometer-sized protein machine, which does not presently exist, will facilitate our comprehensive understanding of many biological processes and diseases, and will accelerate future pharmaceutical regulation of gene expression. To achieve the goal, we will establish a novel experimental approach for single channel recording of the NPC by improving spatial and time resolutions of scanning electrochemical microscope (SECM) to nanometer and microsecond ranges. While SECM is a well-established method for non-invasive and quantitative measurements of membrane permeability, improvement of the spatial resolution by nanofabricated SECM probes will enable us to measure the single NPC permeability. Real-time information about the individual channel events can be obtained by monitoring their effects on the single NPC permeability with high time resolution. The time-resolved information will allow us to evaluate the transport models whose validity can not be checked by currently-available experimental data, leading to more comprehensive understanding of this important transport process.