Single Channel Recording of the Nuclear Pore Complex

描述（由申请人提供）：细胞核是真核细胞的中心细胞器。细胞核和细胞质之间的分子运输仅通过核孔复合物（NPC）介导，该复合物穿过定义核室的双膜核包膜。我们的长期目标是获得单个NPC中单个通道门控和蛋白质易位事件的实时信息。目前尚不存在的通过纳米级蛋白质机器进行核胞质运输的信息，将有助于我们对许多生物过程和疾病的全面了解，并将加速未来基因表达的药物调控。为了实现这一目标，我们将建立一种新的单通道记录NPC的实验方法，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。虽然SECM是一种成熟的非侵入性和定量测量膜通透性的方法，但通过纳米制造的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。通过高时间分辨率监测各通道事件对单个NPC渗透率的影响，可以获得各通道事件的实时信息。时间分辨的信息将使我们能够评估目前可用的实验数据无法验证的传输模型，从而更全面地了解这一重要的传输过程。

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.