A major goal in membrane biology is to understand how transmembrane signals trigger rearrangements of the cytoskeleton that effect cell movement and changes in cell shape. Phagocytosis, a specialized form of endocytosis dependent on the actin-based cytoskeleton and its connections to the plasma membrane, is performed by free-living single-celled organisms such as the soil amoeba Dictyostelium discoideum. This phylogenetically primitive mechanism also is used by specialized ameboid cells (macrophages and leukocytes) of immune systems of animals for host defense as well as for tissue repair and morphogenetic remodeling.
Phagocytosis is a process that requires local alterations in the cytoarchitecture and thus is a controlled remodeling event. The long-term goal of these studies is to understand cytoskeletal-membrane interactions during the formation of the phagocytic cup and during phagosome processing in Dictyostelium. Because D. discoideum amebae perform the same motile activities, such as phagocytosis and chemotaxis, as macrophages and polymorphonuclear leukocytes, the molecular mechanism of phagocytosis in D. discoideum likely will resemble the mechanisms of phagocytes in other cell types, including animal immune systems. Importantly, and in contrast to macrophages and leukocytes, molecular genetic approaches to the study of motile functions such as phagocytosis are feasible in D. discoideum with its haploid and pliable genome.
Previously, a cell-surface glycoprotein gp130 of D. discoideum was identified as a candidate phagocytosis receptor. Based on the amino acid sequence deduced from the recently acquired gene sequence, gp130 is postulated to have an adhesive function and a glycosylphosphatidylinositol (GPI) anchor that links it to the outer leaflet of the plasma membrane of vegetative cells. Although not novel, this GPI anchor raises intriguing questions about the function of gp130 and its interactions with the cytoskeleton. Gp130 is also one of the few identified plasma membrane proteins known to be internalized during phagocytosis and thus it may serve as a useful marker for the endo-lysosomal membrane network in a cell type with high membrane turnover.
Through either gene disruption or antisense methods using the cloned cDNA for gp130, the in vivo function of gp130 will be explored by generating mutant cell lines lacking the gene. The pinocytic and phagocytic activities under different growth conditions, and adhesive properties of gp130-minus cells will be tested and compared to the parent strain. Biochemical analyses, aided by gp130 antibodies, will determine whether gp130 is GPI-linked or an integral membrane protein. The antibodies also will be used to examine the distribution patterns of gp130 during different endocytic activities to determine its fate after internalization. In addition, a role of gp130 in cell-cell adhesion during development will be tested by assaying the ability of gp130-minus cells to aggregate, and with binding assays using purified native protein. To augment localization studies, a green fluorescent protein (GFP)-gp130 fusion protein will be introduced into both normal and cells already containing other spectrally-distinct GFP-cytoskeletal proteins, such as actin, involved in the formation of the phagocytic cup. The ability to monitor gp130 and these other proteins in live cells during phagocytosis will provide temporal and spatial information at the molecular level about this dynamic process that could not be acquired through traditional biochemical and microscopy methods. Finally, actin polymerization kinetics will be measured and compared between normal and gp130-minus cells engaged in phagocytosis to determine if there is a functional linkage between gp130 and the actin cytoskeleton. Because gp130 may be signaling to the cytoskeleton, it likely interacts with other proteins. Future studies will focus on the identification and activities of these associated proteins because they will provide information about the signaling mechanism(s) between cell-surface molecules and the cytoskeletal network responsible for activities, such as cell motility, cell-cell adhesion and cell substrate adhesion that are fundamental to tissue and organ development.
