ABSTRACT
Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.
Subject(s)
Immunohistochemistry/methods , Metal Nanoparticles/chemistry , Staining and Labeling/methods , Actins/metabolism , Antibodies/immunology , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line, Tumor , Cell Nucleus , Gold/chemistry , HeLa Cells , Humans , Microscopy, Electron , Nuclear Proteins/metabolism , Nucleophosmin , Phosphatidylinositol 4,5-Diphosphate/metabolism , Ribonucleoproteins, Small Nuclear/metabolismABSTRACT
A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.
Subject(s)
Cell Nucleus/metabolism , Molecular Motor Proteins , Myosins/metabolism , RNA Polymerase II/metabolism , RNA/biosynthesis , Transcription, Genetic , 3T3 Cells , Actins/metabolism , Amanitins/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Dactinomycin/pharmacology , Exons , HeLa Cells , Humans , Mice , Microscopy, Confocal , Microscopy, Electron , Molecular Sequence Data , Myosins/chemistry , Myosins/genetics , Myosins/immunology , Nucleic Acid Synthesis Inhibitors/pharmacology , Precipitin Tests , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolismABSTRACT
The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.
