CHAPERONIN FILAMENTS ARCHAEAL CYTOSTRUCTURE

(部分由能源部61225-00-110至J.Trent支持) 当今生物学的基本问题是蛋白质如何折叠成它们的 在拥挤的活细胞的范围内发挥功能。近期 研究表明，这一过程是由一类蛋白质介导的 称为分子伴侣。世界上最重要的监护人之一 这一过程是一种称为伴侣的多亚基复合体，是一种 双环结构被认为是将蛋白质折叠到其中空内 核心。我们有来自纯化的伴侣蛋白的证据表明它们聚集在一起 在体外形成有序的细丝，并在细胞中观察到 说明体内也存在类似的细丝。我们建议使用BMIRR 设施到1.查看最低限度处理的冷冻水化细胞内部 或用高压电子显微镜或IVEM进行厚冰冻切片，以改善图像。 通过常规的EM技术在细胞中看到的内部结构； 收集高质量的负片染色和冰冻水合图像 将分子伴侣细丝与细胞中所见的细丝进行比较 澄清它们的结构关系，并深入了解 分子伴侣细丝形成的基础；和3.使用3D 用于对细丝进行成像的断层重建设备 试图将这种结构的性质理解为 用于连接其他单元格组件的位置。在特伦特博士访问期间， 我们用冷冻切割法(Tokuyasu法)检测了柴胡螺旋体细胞。 Hvem，并制作新鲜制备的细胞整装制剂 在文化中成长。录制了许多立体声对，还有一个 断层重建由断层图像重建而成。这个 重建显示S层完好无损，铀酰 醋酸盐污渍没有进入细胞内。在整个坐骑中，我们 可以看穿整个细胞，看到染色的S层， 但我们看不到内部结构。我们还查看了 视频显微镜下培养的细胞。不规则的豆袋形状 可能是由于内部细胞骨架或由于 S层和细胞的膨胀压力。特伦特博士还记录了 几对立体细胞去除了表面层 希望看到伴侣的结构以及它的 建筑。这些图像很难解读。样本 特伦特博士带来的大肠杆菌(L形式)，以及 环境和高温条件，都很高 加压冷冻，总共30次。这些细胞是 冷冻替代和嵌入。这些积木被送到特伦特医生那里 用于剖切。

(Supported in part by DOE 61225-00-110 to J. Trent) One of the fundamental problems in biology today is how proteins fold into their functioning form in the crowded confines of living cells. Recent studied suggest that this process is mediated by a class of proteins called molecular chaperones. One of the most important chaperones in this process is a multi-subunit complex known a chaperonin that is a double-ring structure believed to fold proteins inside its hollow core. We have evidence from purified chaperonins that they aggregate in vitro to form ordered filaments, and observations in cells that suggest similar filaments exist in vivo. We propose to use the BMIRR facilities to 1. Look inside minimally treated frozen hydrated cells or thick cryosections by HVEM or IVEM to improve the images of the internal structures seen in cells by conventional EM techniques; 2. Collect high quality images of negatively stained and frozen hydrated chaperonin filaments to compare to the filaments seen in cells to clarify their structural relationship and to gain insights into the basis for chaperonin filament formation; and 3. Use the 3D tomographic reconstruction facilities to image the filaments themselves in an attempt to understand the nature of this structure as a site for attaching other cell components. During Dr. Trent's visit, we examined cryosectioned (Tokuyasu method) S. shibatae cells with the HVEM, and made fresh-prepared whole-mount preparations of cells growing in culture. Many stereo pairs were recorded, and a tomographic reconstruction was made from a section. The reconstruction revealed that the S-layer is intact and that uranyl acetate stain did not get inside the cell. In the whole mounts, we could see through the whole cell and visualize the stained S-layers, but we could not see the internal structures. We also looked at the cultured cells with video microscopy. The irregular, bean-bag, shape seen may be due to an internal cytoskeleton or to features of the s-layer and the cell's turgor pressure. Dr Trent also recorded several stereo pairs of cells that had the surface layer removed in hopes of seeing the structure of chaperonin as well as its architecture. These images were very difficult to interpret. Samples of E-coli (L-forms), brought by Dr. Trent, as well as live cells at both ambient and high-temperature conditions, were high pressure-frozen for a totoal of 30 runs. The cells were freeze-substituted and embedded. The blocks were sent to Dr. Trent for sectioning.