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， 在维持细胞形态中起主要作用的结构。 在这个项目中，我们将研究结构和组装 肌肉和神经细胞中IF蛋白的特性， 化学和高分辨率形态学技术。 我们将 用化学方法合成一些肽， 序列选自结蛋白的序列， 神经丝（NF）三联体多肽，然后用它们来 研究蛋白质结构域的功能作用， 他们已经有主了。 对于结蛋白，我们将直接使用肽作为 竞争性抑制剂的细丝组装，如测量的 新的荧光检测方法。 同时， 分子分辨率下被捕集产物的图像 将使用快速冷冻，深蚀刻技术， 电镜（EM）。 通过检查子单元是如何 如果我们希望能够融入IF， 了解亚基是如何组装在正常的纤维中的。 多克隆抗血清将针对NF-衍生的 合成肽，从C-末端 三重态的延伸，NF-L，NF-M和NF-H。 的抗体 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 NF交叉桥和定位推定的磷酸化位点 NF-M。 我们还将确定，通过荧光能量转移 实验，IF亚基是否结构极性或非极性， 通过两种超微结构方法， 单向或双向。 为了探索如果 可能比一般情况下更有活力，我们将寻找 体外形成的丝之间结蛋白亚基的交换 并跟踪荧光标记的结蛋白亚基在 显微注射到发育中的心脏和骨骼肌细胞中。 我们的长期目标是绘制各种 IF蛋白分子在细丝结构中的结构域， 最终修改那些功能感兴趣的。 这可以允许 我们将修饰的IF蛋白分子插入细胞骨架中， 一种更好地理解细胞骨架的方法 形态发生 这一层次的知识可能有助于 对心肌和骨骼肌形态发生的认识. 更高层次的组织，从而导致更有效的 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.