Ultrastructure

核心D将进行定性和定量的组织学和超微结构检查，这是支持所有其他项目所必需的。核心不仅将提供各种电子、相对比和共聚焦显微镜技术，而且还将以高质量标准运行，并提供对实验和分子改变结果的专家关键评估。一致和一致地使用高质量图像将允许检测蛋白质-蛋白质相互作用和单个细胞器反应中的细微变化，这些变化要么是基于改变的功能，要么是这种变化的长期结果。很明显，肌纤维对影响兴奋-收缩耦合的突变的整体超微结构反应是相当特定的，并提供了对病因影响的相当深入的了解。在过去的一段时间里，我们已经证明了一种强烈的纤维类型和性别依赖病理，这与功能上的类似变化相当吻合。提出了两种一般的方法。一个是在突变的背景下定义钙释放单元（cru，骨骼肌中的三联体）的主要蛋白质成分之间关系的任何改变，另一个是通过发育和衰老以及与突变对cru通道的已知功能影响相关的病理发展（最具体的是线粒体，肌纤维和cru的改变）。这将通过结合光学显微镜技术（纤维整个支架的相衬和荧光免疫标记纤维的共聚焦成像）与电子显微镜的薄切片和冷冻断裂，辅以定量形态测定技术来实现。该核心旨在确定每种突变在短期内对钙释放单元的大胶质组装的主要影响，以及在长期内对SR、线粒体和收缩物质的次要影响。

Core D will perform the qualitative and quantitative histological and ultrastructural checks that are necessary to support all other Projects. The Core will not only supply various techniques of electron, phase contrast and confocal microscopy, but it will also operate at a high standard of quality and offer an expert critical evaluation of the results of experimental and molecular alterations. The uniform and consistent use of high quality images will allow the detection of even subtle alterations in protein-protein interactions and in the response of individual cell organelles that either are at the basis of altered functions or are the long term results of such alterations. It is clear that the overall ultrastructural response of the muscle fiber to mutations affecting excitation-contraction coupling are quite specific and offer considerable insight into causative effects. In the past period we have evidenced a strong fiber type- and gender-dependence of the pathology, that correspindes quite well with similar variations in function. Two general approaches are proposed. One is to define any alterations in the relationships between the major protein components of calcium release units (CRUs, triads in skeletal muscle) within the context of the mutation and the other is to follow the development of pathology (most specifically mitochondrial, myofibrillar and CRUs' alterations) through development and aging and in relation to the known functional effects of the mutation on CRUs' channels.This will be achieved by combining light microscope techniques (phase contrast of fibers whole mounts and confocal imaging of fluorescently immunolabeled fibers) with thin sectioning and freeze-fracture for electron microscopy supplemented by quantitative morphometry techniques. The core aims at defining the primary impact of each mutation on the macromelcular assembly of calcium release units within a short term and the secondary impact on SR, mitochondria and contractile material on the long term.