The Myosin Family of Dictyostelium

MCB 9507376 Margaret Titus 肌球蛋白家族正在迅速扩大，目前包括九个不同的类，基于系统发育比较。 肌球蛋白的这种多样性引起了关于细胞产生运动所利用的肌动蛋白基马达的性质和程度的有趣问题。 肌球蛋白的最知名的类别，肌球蛋白I，II和V一直是深入研究的主题，然而，关于其他类型的肌球蛋白的体内作用的信息是缺乏的。 在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形式肌动蛋白为基础的运动。 需要一个系统的方法来评估个别类别的肌球蛋白在产生肌动蛋白为基础的运动的作用，探索这种可能性。 我们的工作目标是通过基因打靶的方法来探索单个细胞中不同类型肌球蛋白的体内功能。Dictyosteoblasts是我们的调查的主题，经历了一个简单的，但优雅的发展计划，并表现出类似于在高等真核细胞中观察到的趋化性和运动性。 它适合于分子遗传操作和细胞生物学研究。 五个非常规肌球蛋白先前已确定在这种生物体。 一个新的屏幕，结合物理作图技术与低严格杂交已经确定了至少7个额外的肌球蛋白（肌球蛋白）基因。根据初步的系统发育分析，其中两个myoH和myoJ似乎编码一类新的非常规肌球蛋白的成员。 这些肌球蛋白通过网骨藻的发育周期表达，表明它们在形态发生和发育中发挥作用。 将使用包括细胞生物学和分子遗传学技术的组合方法研究myo H和myoJ基因产物的体内作用。 这些基因将被完全克隆、测序并用于产生融合蛋白。 然后将融合蛋白用于获得用于免疫定位的特异性抗体。 将进行基因靶向研究以产生不表达myoH或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，这些实验将提供深入了解这个潜在的新肌球蛋白家族在产生不同类型的肌动蛋白为基础的细胞运动，在Dictyosteoprotein生命周期中进行的作用。 了解myoH和myoJ基因产物在发育过程中进行的运动中的作用将提供有关细胞如何使用其肌动球蛋白细胞骨架产生各种运动的重要信息。这些可能包括但不限于细胞器移动、细胞器锚定或细胞易位。 这个项目研究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各样的工作。 它们是生命所必需的，并在细胞运动和细胞内成分的运动中起作用。 该项目将导致对这些基本分子的更好理解。 ***

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. ***