Kinetics of binding, uptake and degradation of live fluorescent (DsRed) bacteria by Dictyostelium discoideum.
The kinetics of binding, uptake and degradation of bacteria by vegetative Dictyostelium amoeba using Escherichia coli expressing the recombinant fluorescent protein DsRed have been characterized. There are significant advantages to using DsRed-expressing bacteria for phagocytosis assays. Stable expression of the fluorescent protein, DsRed, provides living bacteria with a bright internal fluorescent signal that is degradable in the phagolysosomal pathway. Unlike assays with chemically labelled bacteria or latex beads, the bacteria are alive and possess a natural, unaltered external surface for receptor interaction. Dictyostelium cells rapidly bind and phagocytose DsRed bacteria. Pulse-chase experiments show that the signal derived from DsRed is degraded with a half-life of approximately 45 min. To distinguish internalized bacteria from those bound to the surface, an assay was developed in which sodium azide was used to release surface-bound particles. Surprisingly, surface particle release appears to be independent of myosin II function. Using this assay it was shown that the uptake of bacteria into cells is extremely rapid. After 1 min incubation, 20% of the signal is derived from internalized bacteria. The proportion of the signal from internalized bacteria increases gradually and reaches 50% at steady state. This assay will be useful in investigations of the molecular machinery of phagocytosis and post-internalization vesicle trafficking.
Maselli
Andrew
A
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA1.
Laevsky
Gary
G
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA1.
Knecht
David A
DA
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA1.
D000818:Animals; D001422:Bacterial Adhesion; D004023:Dictyostelium / Q000382:microbiology* / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D024462:Myosin Type II / Q000502:physiology; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000382:microbiology / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011994:Recombinant Proteins; C413663:fluorescent protein 583; D024462:Myosin Type II