| Mapping of adherens junction components using microscopic resonance energy transfer imaging. | |
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MedLine Citation:
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PMID: 7622593 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions. |
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Authors:
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Z Kam; T Volberg; B Geiger |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Journal of cell science Volume: 108 ( Pt 3) ISSN: 0021-9533 ISO Abbreviation: J. Cell. Sci. Publication Date: 1995 Mar |
Date Detail:
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Created Date: 1995-08-29 Completed Date: 1995-08-29 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0052457 Medline TA: J Cell Sci Country: ENGLAND |
Other Details:
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Languages: eng Pagination: 1051-62 Citation Subset: IM |
Affiliation:
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Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Actinin
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metabolism Actins / metabolism Animals Antibodies, Monoclonal Cell Adhesion / physiology* Cells, Cultured Chickens Energy Transfer* Fluorescein Fluoresceins Humans Image Processing, Computer-Assisted Intercellular Junctions / metabolism Lens, Crystalline / cytology, metabolism Microscopy, Fluorescence / methods* Rabbits Rhodamines Talin / metabolism Vinculin / metabolism |
| Chemical | |
Reg. No./Substance:
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0/Actins; 0/Antibodies, Monoclonal; 0/Fluoresceins; 0/Rhodamines; 0/Talin; 11003-00-2/Actinin; 125361-02-6/Vinculin; 2321-07-5/Fluorescein |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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