ENDOMEMBRANES SYSTEMS OF GIARDIA

贾第虫的内膜系统

• 批准号: 6220677
• 负责人: MARCIA ATTIAS
• 金额: $ 3.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220677
• 关键词: Mammalia; biomedical resource; computers; enzymes; human tissue; microscopy; musculoskeletal system; nervous system; technology /technique

This past year, we finished a seminal study on the structure of neuronal mitochondria from chick and rat using electron microscope tomography of chemically fixed tissue. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with the intermediate voltage electron microscope (400 kV) at NCMIR. We found that the mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ~7 nm thick. The inner boundary membrane was found to lie close to the outer membrane with a total thickness across both membranes of ~22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, ~14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ~14 nm) These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion. Another study that has recently been finished used electron microscope tomography on both cryofixed and chemically fixed brown adipose tissue (BAT) to examine the membrane topology of BAT mitochondria, which possess unique bioenergetics due to an uncoupling protein. The three-dimensional reconstructions of BAT mitochondria provided a view different in important biomembrane features from descriptions found in the literature. We gained new insight into BAT mitochondria architecture by identifying crista junctions, including multiple junctions connecting a crista to the same side of the intermembrane space, in a class of mitochondria that have no tubular cristae, but only lamellar cristae. We found that the cristae architecture of cryofixed mitochondria, including crista junctions, is identical to that found in chemically fixed mitochondria suggesting that this architecture is not a fixation artifact and is likely found in vivo. In cryofixed mitochondria almost all of the outer membrane was observed to be in close contact with the inner boundary membrane, which has implications for the exchange of ATP/ADP across these membranes. The stacks of lamellar cristae extended through more of the BAT mitochondrial volume than did the cristae we observed in neuronal mitochondria. Hence, the inner membrane surface area was larger in the former, which may reflect the additional surface occupied by the uncoupling protein, but may also result from a higher concentration of electron transport proteins. This is consistent with the high metabolic/thermogenic activity of BAT mitochondria. cAMP-dependent protein kinase (PKA), one of the first protein kinases discovered, mediates a variety of hormonal and neurotransmitter responses by phosphorylating different substrate proteins in the cell. Compartmentalization of PKA is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). This past year, we have made significant progress in identifying the physiological partners of PKA and PKC with a selected subset of AKAPs using immunochemical methods coupled with confocal microscopy and electron microscopy Most of this work has been with a novel AKAP, called D-AKAP1 which binds both type I and type II regulatory subunits of PKA. Although PKA is a multifunctional enzyme with a broad substrate specificity, activation of this kinase permits preferential phosphorylation of specific target substrates. While the importance of PKA in regulating many cellular processes has long been apparent, the potential importance of compartmentalization for the function and regulation of PKA has only recently been recognized. Investigation of compartmentalization of PKA by fluorescently labeling the regulatory subunits and D-AKAP1 has been our major endeavor this past year and utilized both laser scanning confocal microscopy and electron microscopy to determine labeling on three levels. The first level is cellular, i.e. to determine which cell type expresses D-AKAP1. The second level is subcellular; which subcellular structures have D-AKAP1. The third level is suborganellar, e.g., does D-AKAP1 bind to the inner or outer mitochondrial membrane. Confocal microscopy is principally used at the first two levels, while electron microscopy is required for the third level. Being able to visualize these anchoring proteins and the physiological PKA and PKC partners in cells is now affording us an understanding of how these molecules function in living cells. In the coming year, we will generate high-resolution 3-D reconstructions of antibody-labeled mitochondra by electron tomography to provide a foundation for the mapping of AKAPs, PKA and PKC in those cells that show mitochondrial labeling. Another project involving the IVEM was completed in 1997. This project looked at RXRa null mutant mice which display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To help dissect the molecular basis of the RXRa-associated cardiomyopathy, we performed ultrastructural studies on sections of embryonic heart which suggested that the density of mitochondria per myocyte was higher in the RXRa mutant compared to wild-type littermates.

在过去的一年中，我们完成了一项关于结构的开创性研究 使用电子显微镜从雏鸡和大鼠的神经元线粒体 化学固定组织的断层扫描。 三维 代表性线粒体的重建是由 用中间电压获得的单轴倾斜系列 NCMIR的电子显微镜（400 kV）。 我们发现 线粒体超微结构在​​物种和神经元之间相似 地区。 外膜和内膜厚约7 nm。 这 发现内部边界膜靠近外膜 两个膜的总厚度约为22 nm。 我们发现了 内膜只能通过 狭窄的管状开口，我们称之为Crista连接。 有时 cristae在整个长度上保持管状，但通常是多个 管状crista合并形成层状室。 点状 观察到直径约14 nm的区域，其中内部和 外膜出现在接触中（两个膜的总厚度 〜14 nm）这些接触站点在 将蛋白质转运到线粒体中。 它一直 假设接触站点可能靠近Crista连接 促进蛋白质的运输，原定为Cristae。 然而， 我们的统计分析表明接触位点是随机的 相对于这些连接。 此外，接近 观察到内质网膜和 线粒体外膜，与报道的 某些脂质进入线粒体的机理。 最近已经完成的另一个研究了电子显微镜 在冷冻和化学固定的棕色脂肪组织上的断层扫描 （BAT）检查蝙蝠线粒体的膜拓扑 由于未偶联蛋白而具有独特的生物能力。 这 蝙蝠线粒体的三维重建提供了视图 重要的生物膜特征与在 文学。 我们对蝙蝠线粒体有了新的见解 通过识别Crista连接的架构，包括多个 将Crista连接到膜的同一侧的连接处 空间，在没有管状cristae的线粒体中 只有层状cristae。 我们发现Cristae的建筑 Cryofixed线粒体（包括Crista连接）与 在化学固定的线粒体中发现的，这表明 建筑不是固定的伪像，很可能在体内发现。 在冰冻的线粒体中，几乎所有的外膜都是 观察到与内边界膜密切接触， 这对在这些方面的交流ATP/ADP具有影响 膜。 层层堆积延伸到更多 蝙蝠线粒体体积比我们在 神经元线粒体。 因此，内膜表面积为 前者更大，这可能反映了额外的表面 被解耦蛋白占据，但也可能是由于较高的 电子转运蛋白的浓度。 这与 蝙蝠线粒体的高代谢/热活性。 cAMP依赖性蛋白激酶（PKA），第一个蛋白激酶之一 发现，介导各种激素和神经递质 通过磷酸化细胞中的不同底物蛋白来反应。 PKA的隔离是通过与 A-激酶锚定蛋白（AKAP）。 过去一年，我们做了 在识别PKA的生理伙伴方面取得了重大进展 PKC使用免疫化学方法具有选定的AKAP子集 与共聚焦显微镜和电子显微镜结合大多数 工作与一种新颖的AKAP，称为d-akap1，绑定了这两种类型 I和PKA的II型监管亚基。 虽然PKA是一个 具有广泛底物特异性的多功能酶激活 这种激酶允许特定靶标的优先磷酸化 基材。 而PKA在调节许多细胞中的重要性 过程长期以来一直很明显，这是潜在的重要性 PKA功能和调节的隔室化仅具有 最近被认可。 调查 PKA通过荧光标记调节亚基，D-AKAP1具有 在过去的一年中一直是我们的主要努力，并利用了两个激光器 扫描共聚焦显微镜和电子显微镜确定 在三个级别上标记。 第一级是细胞，即 确定哪种细胞类型表示D-AKAP1。 第二层是 亚细胞；哪些亚细胞结构具有D-AKAP1。 第三 级别是subganellar，例如，d-akap1与内部或外部结合 线粒体膜。 共聚焦显微镜主要用于 前两个级别，而电子显微镜需要 第三级。 能够可视化这些锚定蛋白和 细胞中的生理PKA和PKC伴侣现在为我们提供了一个 了解这些分子如何在活细胞中起作用。 在 来年，我们将生成高分辨率3-D重建 通过电子断层扫描抗体标记的线粒体，以提供 在这些单元格中映射AKAP，PKA和PKC的基础 显示线粒体标记。 涉及IVEM的另一个项目是 该项目于1997年完成。该项目查看了RXRA NULL突变小鼠 显示眼和心脏畸形，肝发育延迟， 并死于胚胎日（E）14.5 PC周围的心力衰竭。 到 有助于剖析与RXRA相关的分子基础 心肌病，我们对 胚胎心脏，表明线粒体的密度为 与野生型相比，RXRA突变体的肌细胞更高 同窝工人。