Macromolecular Organization and Function of the Aqueous Cytoplasm

这项拟议的研究是一项旨在了解动物细胞水室的结构、性质和代谢活动的长期计划的继续，这些水室包括核质、细胞质膜结合的细胞器内部和胞浆或水胞质。虽然这些隔室约占大多数细胞质量和体积的75%，并且是代谢活动的场所，但人们对它们的性质和组织的细节知之甚少。目前的工作主要集中在细胞质内，主要使用培养的小鼠成纤维细胞(L细胞)，这些细胞已经被硫酸葡聚糖渗透(数字信号处理器细胞)。它们保留了通过更严厉的处理(洗涤剂提取或均质)释放到介质中的大多数大分子：这些大分子通常被认为是自由溶解(“溶解”)在水中的细胞质。这些大分子中的大多数保留在DSP细胞中，尽管它们的质膜中有足够大的损伤(“孔”)，使它们能够逃脱。这项研究旨在检验这样一种假设，即通过提取和经典细胞分级定义的大多数“可溶”大分子实际上在完整的细胞中并不是游离的，而是有组织的，并与细胞基质松散地联系在一起。这一假说将在DSP细胞中通过使用各种代谢探针和高压电子显微镜检查糖酵解的酶(长期以来被认为是“可溶的”)来验证。这些酶(以及一般的蛋白质)从DSP细胞中流出，暴露在旨在扰乱假定的细胞基质-酶关联的处理中，将通过电泳法和酶活性分析进行研究。还将启动对其他类型细胞的DS渗透性的研究。尽管人们对活细胞的细胞质在大分子水平上的组织知之甚少，但长期以来，它一直被忽视为严格研究的对象，因为细胞质通常被认为是蛋白质和小分子的简单溶液。来自细胞生物学和生物化学的几条最新证据表明，这是对细胞的过于简单化和误导性的观点。拟议中的研究将提供关于细胞中蛋白质和小分子组织状态的迫切需要的数据，并可能导致对细胞结构和功能的根本不同和更现实的概念。

The proposed research is the continuation of a long range program aimed at understanding the structure, properties and metabolic activities of the aqueous compartments of animal cells: nucleoplasm, interiors of cytoplasmic membrane-bound organelles, and the cytosol, or aqueous cytoplasm. Although these compartments make up about 75% of the mass and volume of most cells, and are the sites of much metabolic activity, very little is known about the details of their properties and organization. Current work focuses on the aqueous cytoplasm, and employs chiefly cultured mouse fibroblasts (L cells) which have been permeabilized with dextran sulfate (DSP cells). They retain most macromolecules that are released into the medium by harsher treatments (detergent extraction or homogenization): those macromolecules commonly thought to be freely dissolved ("soluble") in the aqueous cytoplasm. Retention of most of these macromolecules in DSP cells occurs in spite of lesions ("pores") in their plasma membranes that are large enough to allow them to escape. This research is designed to test the hypothesis that most "soluble" macromolecules defined by extraction and classical cell fractionation are not actually free in intact cells but are instead organized, and associated loosely with the cytomatrix. The hypothesis will be tested in DSP cells by an examination of the enzymes of glycolysis (long believed to be "soluble") using a variety of metabolic probes and high voltage electron microscopy. The efflux of these enzymes (and proteins in general) from DSP cells exposed to treatments designed to perturb postulated cytomatrix-enzyme associations will be studied by electrophoretic and enzyme activity assays. Studies on DS permeabilization of other cell types will also be initiated. Although little is known about the organization of the cytoplasm of living cells at the macromolecular level, it has long been overlooked as a subject of rigorous study because "cytoplasm" has generally been regarded as a simple solution of proteins and small molecules. Several lines of recent evidence from cell biology and biochemistry suggest that this is an oversimplified and misleading view of the cell. The proposed research will provide much needed data on the state of organization of proteins and small molecules in cells and could lead to a fundamentally different and more realistic concept of cellular structure and function.