NEW PROBE AND METHODS FOR CORRELATED LM AND EM

描述（由申请人提供）：我们建议改进和利用强大的新的遗传编码标记系统，通过电子显微镜（EM）与光学显微镜（LM）相关联来可视化蛋白质。EM是在光学分辨率以下观察细胞结构的最强大的技术之一，但由于缺乏普遍适用的遗传学方法， 直到我们最近开发了miniSOG，一种小黄素蛋白，它对EM的作用就像绿色荧光蛋白对LM的作用一样。我们的目标是量化miniSOG的灵敏度和空间分辨率，并将其适用性扩展到低温样品制备和成像方法。将开发和表征替代的遗传编码标记，以允许通过来自缀合至不同镧系元素的二氨基联苯胺的氧化的反应沉积物的电子能量损失光谱在单个EM图像中区分两种或更多种感兴趣的蛋白质。我们还开发了基于来自丙型肝炎病毒蛋白酶的药物可控的顺式作用蛋白酶的报告基因，以区分感兴趣的遗传特异性蛋白质的旧拷贝和新合成拷贝。这些融合标签是可见的相关的LM和EM，并将被施加到可塑性和疾病相关的突触蛋白，以揭示其本地化的外观和营业额，最初在文化BT最终在完整的哺乳动物大脑。将创建病毒和Cre/lox模块化靶向载体，以使上述EM/LM标签的细胞类型选择性表达稳健并广泛适用于像疾病和学习的小鼠模型一样复杂的系统。我们选择了在肝纤维化和突触可塑性中重要的细胞类型和蛋白质作为测试案例，因为这些生物学特性 过程是多种多样的，涉及优秀的当地合作者，并具有很大的生物医学重要性。我们将与合作者一起研究肝细胞、肝星状细胞和内皮细胞在纤维化损伤过程中如何改变超微结构形态和关键蛋白的位置。另一项合作将集中在恐惧条件反射和记忆巩固过程中杏仁核突触的形态和粘附分子的活动诱导变化。将这些新工具扩展到转基因动物中，将使得将遗传特异性蛋白质的超微结构定位和代谢周转与整个动物行为和疾病联系起来成为可能。

DESCRIPTION (provided by applicant): We propose to refine and exploit powerful new genetically encoded labeling systems to visualize proteins by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM. We aim to quantify the sensitivity and spatial resolution of miniSOG and to extend its applicability to low-temperature methods for sample preparation and imaging. Alternative genetically encoded labels will be developed and characterized to allow for two or more proteins of interest to be distinguished in a single EM image by electron energy loss spectroscopy of reaction deposits from oxidation of diaminobenzidine conjugated to different lanthanides. We have also developed reporters, based on the drug- controllable cis-acting protease from hepatitis C viral protease, to distinguish between old and newly synthesized copies of a genetically specified protein of interest. These fusion tags are visible by correlated LM and EM and will be applied to plasticity and disease-related synaptic proteins to reveal their localized appearance and turnover, initially in culture bt eventually in intact mammalian brain. Viral and Cre/lox modular targeting vectors will be created to make cell-type-selective expression of the above EM/LM tags robust and widely applicable to systems as complex as mouse models of disease and learning. We have chosen cell types and proteins important in liver fibrosis and synaptic plasticity as test cases because these biological processes are diverse, engage outstanding local collaborators, and have great biomedical importance. With collaborators we will investigate how hepatocytes, hepatic stellate cells, and endothelial cells change ultrastructural morphology and location of key proteins during fibrogenic injury. Another collaboration will focus on activity-induced changes in morphology and adhesion molecules at synapses in the amygdala during fear conditioning and memory consolidation. Such extension of these new tools into transgenic animals will make it possible to relate ultrastructural location and metabolic turnover of genetically specified proteins to whole- animal behavior and disease.