Abstract
To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.
Highlights
The blasticidin-resistant cells were incubated for 1 h in the presence of fluorescent latex beads and washed, and nonfluorescent cells were sorted with a fluorescence-activated cell sorter (FACS)
How does inactivation of PHG1 result in decreased adhesiveness of mutant cells? The fact that phagocytosis of various particles is affected differentially in phg1 mutant cells suggests some element of specificity in the function of the Phg1 protein, namely that Phg1 might act as a receptor for the phagocytosis of certain substrates
In a series of very elegant experiments, it was shown that this receptor recognizes highly hydrophilic particles: bacteria or latex beads coated with HL5 components
Summary
Cell Culture and Internalization Assays—Wild-type cells used in this study are DH1–10 cells, a subclone of DH1 cells. Cells were resuspended in a solution of 50 mM Na2HPO4, pH 9.2, 5 mg of rhodamine-isothiocyanate (ICN Biomedicals Inc.) at 2 ϫ 109 cells/ml and incubated for 30 min under mild agitation They were washed twice in SB plus 40 mM NH4Cl, twice in SB, and frozen in aliquots. Cells were washed once in sterile ice-cold electroporation buffer (10 mM NaPO4, pH 6.1, 50 mM sucrose), mixed with 10 g of BamHI-linearized vector and 10 units of DpnII restriction enzyme and electroporated using a Bio-Rad Gene.
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