The Myosin Family of Dictyostelium

吞噬作用是细胞摄取颗粒的过程。它是单细胞生物获取营养的主要途径，在多细胞动物中，它对于清除组织中的碎片和防御入侵的病原体等功能至关重要。吞噬作用分几个独立的步骤进行：颗粒附着在细胞表面；由肌动蛋白微丝组成的细胞骨架元件聚集到下面的质膜的底部；在颗粒周围构建富含肌动蛋白的吞噬“杯”膜；相对应的质膜融合形成完整的膜包体或吞噬体，围绕在质膜下的颗粒周围；内化，即被膜包裹的颗粒向细胞内部移动。新形成的吞噬体的内容物随后与细胞内膜结合的溶解腔室（如溶酶体）融合而降解。吞噬杯形成后颗粒物理内化的机制尚不清楚。在粒子内化过程中，细胞施加了相当大的力，大约10 - 30皮牛顿。所有可用的研究表明，肌凝蛋白（一种移动肌动蛋白微丝的分子马达）负责产生粒子吞噬所需的力。然而，在吞噬作用中起重要作用的特定肌球蛋白的鉴定至今仍缺乏。最近在黏菌盘状盘基钢霉（Dictyostelium disideum）中发现了一种用于吞噬所需的非常规肌球蛋白myoi，这种黏菌依赖于吞噬来获取营养。缺乏肌成骨的细胞对颗粒的吸收减少了70%。这些细胞的吞噬缺陷不是由于细胞无法结合颗粒，也不能归因于肌动蛋白细胞骨架的普遍紊乱。这种缺陷的特异性表明，在吞噬过程中，肌成骨主要负责产生收缩力。有趣的是，myoi是一种VII类肌球蛋白。这个肌球蛋白家族与小鼠和人类的神经感觉功能有关，据推测，它在连接肌动蛋白细胞骨架与质膜或参与内吞运输中发挥作用。myosin VII重链由一个保守的myosin运动结构域、蛋白结构颈部区域的3 - 5个轻链结合基序和尾部区域组成，尾部区域具有预测的短段卷曲结构，随后是MyTH4 （myosin tail homology 4）和talin同源结构域的串联重复。MyTH4和talin同源结构域的功能尚不清楚，但据推测，它们要么是肌球蛋白VII的超分子组织所必需的，要么是与靶分子或调节分子结合所必需的。在Dictyostelium（一种易于分子遗传操作的简单真核生物）中鉴定出VII类肌球蛋白，使其成为对该肌球蛋白进行详细功能分析的理想系统。myoi在吞噬中的作用将首先通过在吞噬过程中对突变细胞的实时观察来分析。将对myoi进行绿色荧光蛋白（GFP）标记的互补研究，并确定其在吞噬过程中的分布，并将其与吞噬过程的不同阶段相关联。位点定向诱变将用于创建尾部结构域的各种元素的缺失，并且表达这些缺失结构的细胞的表型将被分析以确定改变的肌球蛋白的功能和分布。这些实验的结果将提供第一个被发现直接参与吞噬的肌球蛋白的作用特征，并将允许确定尾部区域是否对适当的定位和/或功能至关重要。肌球蛋白家族的运动蛋白在真核细胞中普遍存在，并且长期以来被认为负责从亚细胞成分的细胞内运动到细胞爬行和肌肉收缩等全细胞运动的基本运动功能。近年来，许多不同类型的肌凝蛋白马达已被鉴定并在分子方面进行了表征。然而，这些不同的肌凝蛋白的功能意义仍然未知。这个项目的重点是极少数所谓的“非常规”肌球蛋白类型之一，其独特的功能已经相关。相对简单但遗传上易于处理的黏菌盘状钢霉是进行这些研究的理想生物，因为在这些细胞中有大量关于肌动蛋白、肌球蛋白和相关蛋白的背景信息，并且因为它经历了许多依赖于肌动蛋白和肌球蛋白的基本细胞过程（包括变形体运动和吞噬）。这项工作将有助于更好地理解非常规肌凝蛋白，特别是VII类肌凝蛋白在所有真核细胞中的作用。

Phagocytosis is the process by which cells ingest particles. It is a major means by which unicellular organisms obtain nutrition, and in multicellular animals, it is essential for functions such as the clearance of debris in tissues and defense against invading pathogens. Phagocytosis proceeds in several discrete steps: particle attachment to the surface of the cell; recruitment of cytoskeletal elements consisting of actin microfilaments to the underside of the underlying plasma membrane; construction of a phagocytic "cup" of actin-rich membrane around the particle; fusion of the apposing plasma membranes to form a complete membrane enclosure, or phagosome, around the particle just under the plasma membrane; and internalization, i.e., movement of the membrane-enclosed particle to the interior of the cell. The contents of the newly formed phagosome are then degraded following fusion with intracellular membrane-bound lytic compartments (e.g., lysosomes). The mechanism by which the particle is physically internalized following phagocytic cup formation remains unknown. The cell exerts a significant amount of force during particle internalization, around 10 - 30 piconewtons. All available studies indicate that a myosin (a molecular motor which moves actin microfilaments) is responsible for generating the forces necessary for particle engulfment. However, the identification of a specific myosin that plays an essential role in phagocytosis has until now been lacking. An unconventional myosin required for phagocytosis, myoi, has recently been identified in the slime mold Dictyostelium discoideum, which is dependent on phagocytosis for nutrition. Cells lacking myoi exhibit a 70% decrease in the uptake of particles. The phagocytosis defect in these cells is not due to a failure of the cells to bind the particle, nor can it be attributed to a general disorganization of the actin cytoskeleton. The specificity of the defect suggests that myoi is largely responsible for generating contractile forces during phagocytosis. Interestingly, myoi is a class VII myosin. This family of myosins has been implicated in neurosensory functions in both mice and humans, where it has been speculated to play a role in either linking the actin cytoskeleton to the plasma membrane or participating in endocytic trafficking. The myosin VII heavy chain is comprised of a conserved myosin motor domain, three to five light chain binding motifs in the neck region of the protein structure, and a tail region that has a short stretch of predicted coiled-coil structure followed by a tandem repeat of a MyTH4 (myosin tail homology 4) and talin homology domains. The functions of the MyTH4 and talin homology domains are unknown, but it has been speculated that they are required either for the supramolecular organization of myosin VII or for binding to targeting or regulatory molecules. The identification of a class VII myosin in Dictyostelium, a simple eukaryote amenable to molecular genetic manipulation, makes this an ideal system in which to carry out detailed functional analysis of this myosin. The role of myoi in phagocytosis will be analyzed first by real-time observation of the mutant cells during phagocytosis. Complementation studies will be carried out with Green Fluorescent Protein (GFP) tagged myoi and its distribution during phagocytosis will be determined and correlated with the different stages of this process. Site directed mutagenesis will be used to create deletions of various elements of the tail domain, and the phenotypes of cells expressing these deletion constructs will be analyzed to determine the function and distribution of the altered myosins. The results of these experiments will provide a characterization of the role of the first myosin found to be directly involved in phagocytosis, and will allow a determination as to whether the tail region is essential for proper localization and/or function.The myosin family of motor proteins is ubiquitous among eukaryotic cells, and has long been known to be responsible for fundamental motility functions ranging from intracellular movement of subcellular components to whole cell movements such as cell crawling and muscle contraction. In recent years, many distinctly different types of myosin motors have been identified and characterized in molecular terms. However, the functional signficance of these various myosins has remained unknown. This project focuses on one of the very few so-called "unconventional" myosin types for which a distinct function has been correlated. The relatively simple yet genetically tractable slime mold, Dictyostelium, is an ideal organism for these studies, because there is a great deal of background information known about actin, myosins, and associated proteins in these cells, and because it undergoes many fundamental cellular processes that are dependent on actin and myosin (including ameboid motility and phagocytosis). The work will lead to a better understanding of the role of unconventional myosins, particularly the class VII myosins, in all eukaryotic cells.