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In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial ch...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1978
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2110213/
https://www.ncbi.nlm.nih.gov/pubmed/701373
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collection PubMed
description To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.
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spelling pubmed-21102132008-05-01 In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques J Cell Biol Articles To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible. The Rockefeller University Press 1978-10-01 /pmc/articles/PMC2110213/ /pubmed/701373 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title_full In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title_fullStr In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title_full_unstemmed In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title_short In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
title_sort in vitro fusion of acanthamoeba phagolysosomes. ii quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2110213/
https://www.ncbi.nlm.nih.gov/pubmed/701373