Visualization of acidic organelles in intact cells by electron microscopy

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Abstract

We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

Original languageEnglish (US)
Pages (from-to)4838-4842
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number15 I
StatePublished - 1984

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Organelles
Dinitrophenols
Electron Microscopy
Fibroblasts
Coated Vesicles
Multivesicular Bodies
Monensin
Proton-Motive Force
Rhodamines
Endosomes
Ionophores
Chloroquine
Horseradish Peroxidase
Lysosomes
Aldehydes
Anti-Idiotypic Antibodies
Monoclonal Antibodies
Rabbits
Light
Membranes

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "Visualization of acidic organelles in intact cells by electron microscopy",
abstract = "We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.",
author = "Anderson, {R. G W} and Falck, {J. R.} and Goldstein, {J. L.} and Brown, {M. S.}",
year = "1984",
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T1 - Visualization of acidic organelles in intact cells by electron microscopy

AU - Anderson, R. G W

AU - Falck, J. R.

AU - Goldstein, J. L.

AU - Brown, M. S.

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N2 - We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

AB - We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

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