STRUCTURE AND ASSEMBLY OF INTERMEDIATE FILAMENTS

中间丝(IF)、微丝、微管和 它们的辅助蛋白组成了细胞骨架，这是一种复合体 在维持细胞形态方面起主要作用的结构。 在这个项目中，我们将检查结构和组装 肌肉和神经细胞中IF蛋白的特性 化学和高分辨率形态技术。我们会 用化学方法合成多个氨基酸组成的多肽 序列选自desmin和 神经丝(NF)三联体多肽，然后用它们 研究蛋白质结构域的功能作用 他们已经被带走了。对于结蛋白，我们将直接使用这些多肽作为 细丝组装的竞争性抑制物，通过A测量 我们开发的新的荧光分析方法。伴随而来的是， 被截获的组装产品的分子分辨率图像 将使用快速冷冻、深度蚀刻技术来产生 电子显微镜(EM)。通过检查亚基是如何 如果我们希望能够 了解亚基是如何包装在正常的细丝中的。 将产生针对核因子来源的多克隆抗血清 合成肽，取自C-末端 三联体的扩展，即NF-L、NF-M和NF-H。抗体 将用于免疫印迹和免疫标记实验 为了检验C-末端延伸构成 核因子交叉桥和定位假定的磷酸化位点 核因子-M。我们还将通过荧光能量转移来确定 实验，无论亚基在结构上是极性的还是非极性的， 如果它们组装在一起，可以用两种超微结构方法 单向或双向。探索这样一种观念，即如果 可能比通常情况下更具活力，但我们将寻找 结蛋白亚基在体外形成的纤维间的交换 并跟随荧光标记的结蛋白亚基的命运 对发育中的心肌和骨骼肌细胞进行显微注射。 我们的长期目标是绘制出不同的 丝状结构中IF蛋白分子的结构域和 最终修改那些功能上感兴趣的。这可能会允许 美国将把修饰的IF蛋白分子插入细胞骨架作为 一种更好地了解细胞骨架的方法 形态发生。这一水平的知识可能有助于 对心肌和骨骼肌形态发生的理解 更高级别的组织，从而带来更高的效率 预防心脏和其他肌肉疾病。

Intermediate filaments (IF), microfilaments, microtubules and their accessory proteins make up the cytoskeleton, a complex structure that plays a major role in the maintenance of cell form. In this project, we will examine the structure and assembly properties of the IF proteins in muscle and nerve cells using chemical and high resolution morphological techniques. We will chemically synthesize a number of peptides whose amino acid sequences are selected from the sequences of desmin and the neurofilament (NF) triplet polypeptides and then use them to examine the functional role of the protein domains from which they are taken. For desmin, we will use the peptides directly as competitive inhibitors of filament assembly, as measured by a new fluorescence assay that we have developed. Concomitantly, images of the arrested assembly products at molecular resolution will be generated using the quick-freeze, deep-etch technique for electron microscopy (EM). By examining how the subunits are prevented from fitting into the IF we hope to be able to understand how the subunits are packed in a normal filament. Polyclonal antisera will be generated against the NF-derived synthetic peptides, which are taken from the C-terminal extensions of the triplet, NF-L, NF-M and NF-H. The antibodies will be used in immunoblotting and immunolabeling experiments to test the hypothesis that the C-terminal extensions make up the NF crossbridges and to localize the putative phosphorylation site of NF-M. We will also determine, by fluorescence energy transfer experiments, whether IF subunits are structurally polar or apolar, and by two ultrastructural approaches if they assemble unidirectionally or bidirectionally. To explore the notion that IF may be more dynamic than commonly though, we will look for exchange of desmin subunits between formed filaments in vitro and follow the fate of fluorescently labeled desmin subunits after microinjection into developing cardiac and skeletal muscle cells. Our long term goal is to map the roles played by the various domains of the IF protein molecule in filament structure and eventually to modify those of functional interest. This may allow us to insert modified IF protein molecules into the cytoskeleton as a means to gain better understanding of cytoskeletal morphogenesis. Knowledge at this level may contribute to the understanding of cardiac and skeletal muscle morphogenesis at higher levels of organization and thus lead to more effective prevention of heart and other muscle diseases.