The Myosin Family of Dictyostelium

盘基网柄菌的肌球蛋白家族

• 批准号: 9507376
• 负责人: Margaret Titus
• 金额: $ 27万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507376&HistoricalAwards=false
• 关键词: Myosin; Family; Dictyostelium

MCB 9507376 Margaret Titus The myosin family is rapidly expanding and currently consists of nine distinct classes, based on phylogenetic comparisons. This variety of myosins has raised interesting questions about the nature and extent of the actin-based motors utilized by the cell to generate movements. The best known classes of myosins, myosins I, II and V have been the subject of intense study, however information regarding the in vivo role of other types of myosins is lacking. The structural diversity in the myosin family suggests that each class of myosin generates a unique form of actin-based motility. A systematic approach to assessing the role of individual classes of myosins in generating actin-based movements is required to explore this possibility. The goal of our work is to explore the in vivo function of the different classes of myosin in a single cell by gene targeting. Dictyostelium is the subject of our investigations as undergoes a simple, yet elegant developmental program and exhibits chemotaxis and motility similar to that observed in higher eukaryotic cells. It is amenable to molecular genetic manipulation and cell biological investigations. Five unconventional myosins have been previously identified in this organism. A novel screen that combines physical mapping techniques with low stringency hybridization has identified at least seven additional myosin (myo) genes. Two of these, myoH and myoJ, appear to encode members of a new class of unconventional myosin, based on preliminary phylogenetic analyses. These myosins are expressed through the developmental cycle of Dictyostelium, suggesting that they play roles in morphogenesis and development. The in vivo role of the myo H and myoJ gene products, will be investigated using a combined approach that encompasses both cell biological and molecular genetic techniques. The genes will be fully cloned, sequenced, and used to generate fusion proteins. The fusion proteins will then be used to obtain specific antibodies for immunolocalization. Gene targeting studies will be carried out to generate strains that do not express either myoH or myoJ, and the mutants carefully analyzed for defects in various aspects of motility and development. Together, these experiments will provide insights into the role of this potentially new myosin family in generating different types of actin-based cellular movements that are carried out during the Dictyostelium life cycle. Understanding the role of myoH and myoJ gene products in movement carried out during development will provide important information about how cells use their actomyosin cytoskeleton to generate various movements. These potentially include, but are not limited to, organelle movement, the anchoring of organelles, or cellular translocation. %%% This project examine a class of molecules referred to as nonconventional myosins. These molecules have only recently been discovered, and they do a variety of types of work within the cell. They are essential for life and act in cell motility and the movement of components within cells. This project will result in a better understanding of these essential molecules. ***

MCB 9507376玛格丽特·蒂图斯肌球蛋白家族正在迅速扩大，根据系统发育比较，目前由九个不同的类别组成。肌球蛋白的这种多样性引发了关于细胞用来产生运动的基于肌动蛋白的马达的性质和范围的有趣问题。肌球蛋白中最著名的几类，肌球蛋白I、肌球蛋白II和肌球蛋白V一直是深入研究的对象，然而，关于其他类型的肌球蛋白在体内的作用的信息缺乏。肌球蛋白家族的结构多样性表明，每一类肌球蛋白产生一种独特的基于肌动蛋白的运动形式。为了探索这种可能性，需要一种系统的方法来评估个别类别的肌球蛋白在产生基于肌动蛋白的运动中的作用。我们工作的目标是通过基因打靶来探索不同类别的肌球蛋白在单个细胞中的体内功能。网柄网柄菌是我们研究的对象，因为它经历了一个简单而优雅的发育程序，并表现出与高等真核细胞中观察到的相似的趋化性和运动性。它适用于分子遗传操作和细胞生物学研究。此前已在该生物体中鉴定出五种非常规肌球蛋白。一种结合物理作图技术和低严格性杂交的新筛查已经识别出至少七个额外的肌球蛋白(Myo)基因。根据初步的系统发育分析，其中两个，myoH和myoJ，似乎编码了一类新的非传统肌球蛋白的成员。这些肌球蛋白在网柄网柄菌的发育周期中都有表达，提示它们在形态发生和发育中发挥作用。将使用结合细胞生物学和分子遗传学技术的方法来研究myo H和myoJ基因产物在体内的作用。这些基因将被完全克隆、测序，并用于产生融合蛋白。然后，融合蛋白将被用来获得特定的抗体，用于免疫定位。将进行基因打靶研究，以产生既不表达myoH也不表达myoJ的菌株，并仔细分析突变体在运动和发育的各个方面的缺陷。总之，这些实验将提供对这个潜在的新肌球蛋白家族在产生不同类型的基于肌动蛋白的细胞运动中的作用的洞察，这些运动在网柄基菌的生命周期中进行。了解myoH和myoJ基因产物在发育过程中运动中的作用，将为细胞如何利用其肌球蛋白细胞骨架产生各种运动提供重要信息。这些可能包括但不限于细胞器的运动、细胞器的锚定或细胞移位。这个项目研究了一类被称为非常规肌球蛋白的分子。这些分子最近才被发现，它们在细胞内做各种类型的工作。它们对生命是必不可少的，并在细胞运动和细胞内成分的运动中发挥作用。这个项目将使人们更好地了解这些重要的分子。***