The Myosin Family of Dictyostelium

吞噬作用是细胞摄取颗粒的过程。 它是单细胞生物获得营养的主要手段，在多细胞动物中，它对于清除组织中的碎片和防御入侵病原体等功能至关重要。 吞噬作用以几个不连续的步骤进行：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下侧;在颗粒周围构建富含肌动蛋白的膜的吞噬“杯”;并置质膜融合以形成完整的膜封闭物，或吞噬体，围绕在质膜下的颗粒;和内在化，即，膜包裹的颗粒向细胞内部的运动。 然后，新形成的吞噬体的内容物在与细胞内膜结合的裂解区室（例如，溶酶体）。 吞噬杯形成后颗粒被物理内化的机制仍然未知。 细胞在颗粒内化期间施加显著量的力，约10 - 30皮牛顿。 所有现有的研究表明，肌球蛋白（一种移动肌动蛋白微丝的分子马达）负责产生颗粒吞噬所需的力。 然而，到目前为止，还没有鉴定出在吞噬作用中起重要作用的特定肌球蛋白。 一种非常规的肌球蛋白所需的吞噬作用，myoi，最近已被确定在黏菌Dictyosteelium discoideum，这是依赖于吞噬营养。 缺乏myoi的细胞表现出颗粒摄取减少70%。 这些细胞中的吞噬缺陷不是由于细胞不能结合颗粒，也不能归因于肌动蛋白细胞骨架的一般性解体。 缺陷的特异性表明，myoi主要负责在吞噬过程中产生收缩力。 有趣的是，Myoi是一种VII类肌球蛋白。 该肌球蛋白家族与小鼠和人类的神经感觉功能有关，推测其在将肌动蛋白细胞骨架连接至质膜或参与内吞运输中发挥作用。 肌球蛋白VII重链由保守的肌球蛋白马达结构域、蛋白质结构颈部区域中的三至五个轻链结合基序和尾部区域组成，尾部区域具有预测卷曲螺旋结构的短延伸，随后是MyTH 4（肌球蛋白尾部同源性4）和talin同源结构域的串联重复。 MyTH 4和talin同源结构域的功能尚不清楚，但据推测，它们是肌球蛋白VII的超分子组织或与靶向或调节分子结合所必需的。 在Dictyosteobacterium，一个简单的真核生物服从分子遗传操作的第七类肌球蛋白的鉴定，使这一理想的系统中进行详细的功能分析，这种肌球蛋白。 myoi在吞噬作用中的作用将首先通过在吞噬作用期间实时观察突变细胞来分析。 将用绿色荧光蛋白（GFP）标记的myoi进行互补研究，并确定其在吞噬作用期间的分布并与该过程的不同阶段相关联。 定点诱变将用于产生尾域的各种元件的缺失，并且将分析表达这些缺失构建体的细胞的表型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被发现直接参与吞噬作用的第一个肌球蛋白的作用的表征，并且将允许确定尾区是否对于适当的定位和/或功能是必需的。并且长期以来已知其负责从亚细胞组分的细胞内运动到整个细胞运动如细胞爬行的基本运动功能，肌肉收缩。 近年来，许多明显不同类型的肌球蛋白马达已被确定和分子方面的特点。 然而，这些不同的肌球蛋白的功能意义仍然未知。 该项目的重点是极少数所谓的“非常规”肌球蛋白类型之一，其中一个独特的功能已经相关。 相对简单但遗传上易于处理的黏菌，网柄霉，是这些研究的理想生物体，因为有大量关于肌动蛋白、肌球蛋白和这些细胞中相关蛋白的背景信息，并且因为它经历许多依赖于肌动蛋白和肌球蛋白的基本细胞过程（包括阿米巴运动和吞噬作用）。 这项工作将导致更好地了解非常规肌球蛋白，特别是VII类肌球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.