Nuclear Pore Complex in Yeast - the Role of FG-repeats in Structure and Transport.

基因包含并组织在细胞核中，细胞核与细胞的其余部分被膜性核膜隔开。细胞核通过核膜中称为核孔复合体(NPC)的通道与细胞的其余部分进行通信。核祖细胞在控制核功能方面起着关键作用，如基因表达和DNA复制。它们是巨大的、高度复杂的蛋白质结构。通过它们的分子必须由运输蛋白携带。我们对不同的运输载体以及它们如何与它们的货物和它们的控制蛋白相互作用有很好的了解。然而，尽管我们确定了组成NPC的大多数蛋白质，并在一定程度上了解了它的结构，但我们不知道运输载体及其货物是如何通过NPC通道的。其中一个原因是鼻咽癌的结构主要是利用两栖类卵母细胞的核膜来确定的，这种核膜适合于电子显微镜(EM)研究，但很难在实验上操作。然而，像酵母这样的模式生物，在那里很容易突变NPC蛋白，并研究出它们在运输中的作用，还没有进行结构研究。因此，我们开发了一些先进的高分辨率成像方法，旨在确定酵母npc的结构。我们与美国的一个小组合作，该小组拥有一套全面的酵母细胞，其中不同组合的NPC蛋白基因发生了突变。这些突变对不同类型货物的运输有特定的影响。我们拥有一种独特的设备，可以使用各种EM方法确定大型蛋白质复合体的3D结构。这包括以纳米级分辨率观察鼻咽癌表面的高分辨率扫描EM，在那里我们可以检测到单个蛋白质。我们可以将抗体连接到小的金标记物上，并使用这些标记来定位结构中的特定蛋白质。我们将使用透射电子显微镜通过‘EM断层扫描’来确定NPC的三维结构，并可以使用抗体金标记法来定位NPC蛋白。我们将使用这些方法来确定鼻咽癌蛋白的突变对鼻咽癌结构的影响。特别是，我们将研究一组已知对运输至关重要并直接参与运输的蛋白质。我们的合作者还构建了表达货物分子的基因，这些分子带有一种名为GFP的荧光蛋白。GFP标签允许通过荧光显微镜在活细胞中跟踪货物分子，但也为通过抗体金标记法识别EM中的蛋白质提供了一个方便的标签。因此，我们可以通过全国人大跟踪货物分子的进展。我们的计划是跟踪特定货物通过NPC的运输路线，看看突变如何影响这条路线。这样的实验将告诉我们移除特定蛋白质的特定部分对NPC结构有什么影响，并将告诉我们这些蛋白质如何对NPC结构做出贡献。我们将研究与不同货物运输有关的基本蛋白质。然后我们将看到这些特定货物与NPC的相互作用是如何改变的，以及它们的运输是如何受到影响的。在过去的10年里，我们对核运输的了解取得了显著的进步，我们了解了运输载体如何与货物相互作用，以及如何精细地控制蛋白质。此外，还发现了运输不同类型货物所涉及的相关路径网络，并对其进行了描述。所有这些路径都汇聚在全国人大上。然而，全国人大仍然有点像一个“黑匣子”。我们知道它是由什么组成的，但我们不知道这些组件是如何组装在一起的，也不知道它们在运输过程中如何与运输载体相互作用。这里提出的工作解决了这一问题，并可能在理解这一关键细胞过程的关键部分如何发生方面取得突破。

Genes are contained and organised in the nucleus which is separated from the rest of the cell by the membranous nuclear envelope. The nucleus communicates with the rest of the cell through channels in the nuclear envelope called nuclear pore complexes (NPCs). The NPCs have a pivotal role in controlling nuclear functions such as expression of genes and replication of DNA. They are massive, highly complex protein structures. Molecules that travel through them have to be carried by transport proteins. We have a good knowledge of the different transport carriers and how they interact with their cargoes and their control proteins. However, despite identifying most of the proteins that make up the NPC and understanding its architecture to a certain degree we do not know how transport carriers and their cargoes are propelled through the NPC channel. One reason for this is that NPC structure has mostly been determined using amphibian oocyte nuclear envelopes which are suited for electron microscopy (EM) studies but are difficult to manipulate experimentally. However model organisms such as yeast where it is easy to mutate NPC proteins and work out there role in transport have not been accessible to structural studies. Therefore we have developed a number of advanced high resolution imaging methods aimed at determining the structure of yeast NPCs. We have collaborated with a group in the USA which has a comprehensive collection of yeast cells where different combinations of NPC protein genes have been mutated. These mutations have specific effects on transport of different types of cargoes. We have a unique facility for determining the 3D structure of large protein complexes using various EM methods. This includes high resolution scanning EM for looking at the surface of the NPC at nanoscale resolution where we can detect individual proteins. We can link antibodies to small gold markers and use these to locate specific proteins in the structure. We will use transmission EM to determine the 3D structure of the NPC by 'EM tomography' and can use antibody-gold labelling to locate NPC proteins. We will use these methods to determine what effect mutations of the NPC proteins have on NPC structure. In particular we will look at a group of proteins that are known to be essential for and directly involved in transport. Our collaborators have also constructed genes expressing cargo molecules that are tagged with a fluorescent protein called GFP. The GFP tag allows the cargo molecules to be followed in live cells by fluorescence light microscopy but also provides a convenient tag for identifying the protein in the EM by antibody-gold labelling. Therefore we can follow the progress of cargo molecules through the NPC. Our plan is to follow the route of transport of a particular cargo through the NPC and see how the mutations affect this route. Such experiments will tell us what effect removing particular parts of specific proteins has on the structure of the NPC and will tell us how these proteins contribute to NPC structure. We will look at essential proteins involved in the transport of different cargoes. We will then see how the interaction of these specific cargoes with the NPC is altered and how their transport is affected. In the past 10 years our understanding of nuclear transport has made phenomenal progress and we understand how transport carriers interact with cargoes and control proteins exquisitely. Moreover a network of related pathways involved in transporting different types of cargoes have been discovered and characterised. All these pathways converge on the NPC. The NPC however remains a bit of a 'black box'. We know what it is composed of, but we don't know how the components fit together or how they interact with the transport carriers during transport. The work proposed here addresses this and could provide a breakthrough in understanding how this pivotal part of a key cellular process occurs.