Actomyosin in a Basal Body-Associated Fibrillar Complex

在纤毛细胞中，基体被包含在独立的丝状笼子中，与基体微管相连。最近的一项研究表明，肌动蛋白是四膜虫基础体笼复合体的一个组成部分，可能是与这些细胞器相关的肌球蛋白系统的一部分(Hoey和Gavin，J.Cell)。科学，第103页，1992年11月)。这个项目有三个主要目标。第一步是确定四体基体笼复合体中肌球蛋白系统的肌球蛋白成分；第二步是确定肌球蛋白在复合体中的定位；第三步是确定基体笼肌球蛋白是否介导肌动蛋白细丝运动。将筛选一种抗口腔器械抗血清与免疫印迹上的肌球蛋白I和肌球蛋白II发生交叉反应。与肌球蛋白多肽交叉反应的抗体将被亲和纯化，并使用免疫金标记法定位四膜虫基础体笼复合体的肌球蛋白样组分。为了显示肌动蛋白细丝的相互作用，将在体外组装的肌动蛋白细丝与纯化的基础体笼状肌球蛋白混合，直接应用于碳涂层网格，负染，并在电子显微镜下观察。真核细胞最重要的运动形式之一是由轴丝搏动介导的，即高度调控的含有微管的细长细胞伸展(纤毛或鞭毛)的来回运动。根据环境的不同，这种运动使细胞能够以调节的方式游泳或在其附近产生局部电流，从而导致邻近环境相对于细胞的运动。纤毛或鞭毛运动的具体例子包括：哺乳动物精子游向卵子，这是哺乳动物繁殖的关键过程；单细胞藻类或原生动物在池水中游泳；通过将含有食物的池水扫入原生动物的口腔来摄食原生动物；以及通过气管和支气管衬里的上皮细胞的纤毛跳动从肺部清除呼吸道粘液。这种细胞的轴丝植根于被称为基体的细胞结构中，这种结构与中心体同源，中心体是细胞内微管阵列(如有丝分裂纺锤体)的根基。尽管对基底的研究已有一个多世纪，但有关其结构、化学、形成和功能的重要问题仍未得到解答。这些细胞器与细胞中的各种纤维复合体形成一定的结构联系，其中之一是笼状物。这些纤维状复合体的结构与功能之间的关系还没有引起研究者的太多关注。最近的研究表明，肌动蛋白存在于纤毛原生动物四膜虫(一种研究睫毛功能的有用模型系统)的基体笼复合体中，根据肌动蛋白的存在，有人提出，有一种肌球蛋白收缩机制参与了对基体位(以及运动方向)的调节。这项建议的重点是鉴定可能的肌球蛋白成分。这种模型的含义远远超出了原生动物的运动性。

In ciliated cells, basal bodies are contained within separate, filamentous cages connected to basal body microtubules. A recent study showed that actin is a component of the basal body cage complex in Tetrahymena and may be part of an actomyosin system associated with these organelles (Hoey and Gavin, J. Cell. Sci., 103, November 1992). There are three main objectives to this project. The first is to identify the myosin component of the putative actomyosin system in the Tetrahumena basal body cage complex; the second is to localize myosin within the complex; and the third is to determine whether basal body cage myosin mediates actin filament motility. An anti-oral apparatus anti-serum will be screened for cross-reactivity with myosins I and II on immunoblots. Antibodies that cross-react with myosin polypeptides will be affinity-purified and used to localize myosin-like components of the Tetrahymena basal body cage complex using immunogold labeling. To show actin filament interactions, in vitro assembled actin filaments will be mixed with purified basal body cage myosin and applied directly to a carbon-coated grid, negatively stained, and viewed by electron microscopy. %%% One of the most important forms of eukaryotic cellular motility is that mediated by axonemal beating: i.e., the highly regulated back- and-forth movement of slender, microtubule-containing cellular extensions (cilia or flagella). Depending on circumstances, such movements enable a cell to either swim in a regulated manner or to create local currents in its vicinity resulting in the movement of the immediate environment with respect to the cell. Specific cases of ciliary or flagellar motility include such diverse examples as: swimming of mammalian sperm toward the ovum, a process critical to mammalian reproduction; swimming of unicellular algae or protozoans in pondwater; feeding of protozoans by sweepinging food-containing pondwater into their oral cavities; and clearance of respiratory mucus from the lungs by the ciliary beat of epithelial cells lining the trachea and bronchi. The axonemes of such cells are rooted in cellular structures called basal bodies, which are homologous to centrosomes, structures in which intracellular microtubular arrays (such as mitotic spindles) are rooted. Although basal bodies have been studied for more than a century, important questions about their structure, chemistry, formation, and function remain unanswered. These organelles form definite structural associations with various fibrillar complexes in the cell, one of which is the cage. The structure-function relationship of these fibrillar complexes has not received much attention from investigators. Recent work has shown actin to be present in the basal body cage complex of a ciliated protozoan, Tetrahymena (a useful model system for studying ciliary function), and on the basis of the presence of actin it was proposed that there is an actomyosin contractile machinery involved in the regulation of basal body position (and by implication, the direction of the movement). This proposal focuses on the identification of the putative myosin component. The implications of such a model go well beyond motility in protozoans.