Overview  -  Phagocytosis is a specialized form of endocytosis typically defined as the internalization of particles 0.5 µm to 1 µm or larger in diameter. It is how many protists feed and vital for the function of immune cells of metazoans. My research program is focused on the phagocytosis process in Dictyostelium , a soil amoeba. These cells are motile and their phagocytic behavior is analogous to the behavior of 'professional phagocytes' such as macrophages and polymorphonuclear leukocytes. Using D. discoideum as an experimental system has a number of well-known advantages. Its relatively inexpensive and ease of growth in the laboratory allows biochemical analyses of molecules. Molecular genetic manipulations are possible, and with the small haploid genome, specific mutants can be produced and characterized. A web site with more information on Dictyostelium is at http://dictybase.org/dicty.html. Three areas of study in the lab are directly related to the three major phases of the phagocytic process. Our long term goals are to understand how: Project 1: food particles are recognized and bound; Project 2: contact with a food particle initiates the formation of a phagocytic cup; Project 3: the food particle, now in a vesicle called a phagosome, is digested and how the phagosomal membrane proteins are presumably recycled. In pursuing these goals, we have used primarily biochemical and immunological strategies in cell physiology studies to examine the phagocytosis process in D. discoideum. More recently, we are using molecular genetic and cytological techniques to address the function of specific proteins that we have identified. Project 1 - Recognition & adhesion Biochemical analyses have implicated the plasma membrane glycoprotein gp130 in the phagocytosis process. As a cell-surface molecule, it may have a role in the recognition or binding of bacterial food particles (1, 2). With the gene for gp130 cloned (3), a several strategies aimed at examining the function of gp130 are possible. These include constructing disruption mutants, and versions that lack glycosylation sites. Also, the fate of gp130 after its internalization will be addressed with green fluorescent protein (GFP) variants so that its movement through the endocytic network can be monitored by video microscopy in real time. We also have a collection of monoclonal antibodies (unpublished) that were raised against phagosomal membrane proteins. Through reverse genetics, these antibodies will aid in identifying additional molecules on the cell -surface that are involved in an early step of phagocytosis. Relevant articles 1. Rezabek, B.L., J. Rodriguez-Paris, J.A. Cardelli, and C.P. Chia. 1997. Phagosomal proteins of Dictyostelium discoideum.  Journal of Eukaryotic Microbiology. 44:284-292. 2. Chia, C.P. 1996. A 130-kDa plasma membrane glycoprotein involved in Dictyostelium phagocytosis.  Experimental Cell Research. 227:182-189. 3. C.P. Chia and LaRosa, P.C. (in preparation) Molecular cloning of Dictyostelium discoideum glycoprotein gp130: a lipid-anchored protein. Project 2 - Actin recruitment & phagosome formation The ingestion phase of phagocytosis is dependent on the actin-based cytoskeleton (1). The recruitment and subsequent depolymerization of actin at the phagosome membrane is co-ordinated by actin-binding proteins (2). The signals responsible for the localized actin recruitment and subsequent dissolution are actively studied by many laboratories. Post-doctoral scientist Dr. Yuan showed that calcium also plays a role (2), and we seek the site (protein or compartment) of calcium action. Relevant articles 1. Rezabek, B.L., J. Rodriguez-Paris, J.A. Cardelli, and C.P. Chia. 1997. Phagosomal proteins of Dictyostelium discoideum.  Journal of Eukaryotic Microbiology. 44:284-292. 2. Yuan, A., and C.P. Chia. 1999. Co-loss of profilin I, II and cofilin with actin from maturing phagosomes of Dictyostelium discoideum.  Protoplasma. 209:214-225. 3. Yuan, A., C.-H. Siu, and C.P. Chia. 2001. Calcium requirement for efficient phagocytosis by Dictyostelium discoideum.  Cell Calcium. 29:229-238. Project 3 - Digestion & processing of phagosomes For D. discoideum, phagosomes with ingested bacteria fuse with lysosomes that contain digestive enzymes. Found in lysosomes, gp70 is an esterase (1) that is recruited to phagosomes (2). Gp70 is one of two proteins found concentrated in crystalline bodies (3) that are associated with the detergent-insoluble cytoskeleton (1). A possible substrate for gp70 may be some form of the lipopolysaccharide molecule (also known as endotoxin) that makes up the cell wall of gram negative bacteria, which contain fatty acids esterified to the lipid A moiety. Relevant articles 1. Chia, C.P., L. Bomblies, and K.K. Taylor. 1998. Cytoskeletal association of an esterase in Dictyostelium discoideum.  Experimental Cell Research. 244:340-348. 2. Yuan, A., and C.P. Chia. 2000. Role of esterase gp70 and its influence on growth and development of Dictyostelium discoideum.  Experimental Cell Research. 261:336-347. 3. Bomblies, L., E. Biegelmann, V. Doring, G. Gerisch, H. Krafft-Czepa, A.A. Noegel, M. Schleicher, and B.M. Humbel. 1990. Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases.  Journal of Cell Biology. 110:669-679.