Document Detail


Kinetics of binding, uptake and degradation of live fluorescent (DsRed) bacteria by Dictyostelium discoideum.
MedLine Citation:
PMID:  11832505     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Andrew Maselli; Gary Laevsky; David A Knecht
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Microbiology (Reading, England)     Volume:  148     ISSN:  1350-0872     ISO Abbreviation:  Microbiology (Reading, Engl.)     Publication Date:  2002 Feb 
Date Detail:
Created Date:  2002-02-08     Completed Date:  2002-04-25     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  9430468     Medline TA:  Microbiology     Country:  England    
Other Details:
Languages:  eng     Pagination:  413-20     Citation Subset:  IM    
Affiliation:
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Bacterial Adhesion
Dictyostelium / microbiology*,  physiology*
Escherichia coli / genetics
Gene Expression
Kinetics
Luminescent Proteins / genetics,  metabolism
Microscopy, Confocal
Myosin Type II / physiology
Phagocytosis / physiology*
Phagosomes / microbiology,  physiology
Recombinant Proteins / genetics,  metabolism
Grant Support
ID/Acronym/Agency:
GM-40599/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 0/Recombinant Proteins; 0/fluorescent protein 583; EC 3.6.1.-/Myosin Type II

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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