The Myosin Family of Dictyostelium

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.