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.
journal_name
Proc Natl Acad Sci U S Aauthors
Anderson RG,Falck JR,Goldstein JL,Brown MSdoi
10.1073/pnas.81.15.4838subject
Has Abstractpub_date
1984-08-01 00:00:00pages
4838-42issue
15eissn
0027-8424issn
1091-6490journal_volume
81pub_type
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