Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes
<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFP...
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| Format: | Article |
| Language: | English |
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BMC
2010-02-01
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| Series: | BMC Cell Biology |
| Online Access: | http://www.biomedcentral.com/1471-2121/11/15 |
| _version_ | 1818665366982754304 |
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| author | Buckheit Robert W Baker Susan M Falk Matthias M |
| author_facet | Buckheit Robert W Baker Susan M Falk Matthias M |
| author_sort | Buckheit Robert W |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p> |
| first_indexed | 2024-12-17T05:47:30Z |
| format | Article |
| id | doaj.art-5e842c26cdd74a3abe5495395a3710f8 |
| institution | Directory Open Access Journal |
| issn | 1471-2121 |
| language | English |
| last_indexed | 2024-12-17T05:47:30Z |
| publishDate | 2010-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cell Biology |
| spelling | doaj.art-5e842c26cdd74a3abe5495395a3710f82022-12-21T22:01:16ZengBMCBMC Cell Biology1471-21212010-02-011111510.1186/1471-2121-11-15Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopesBuckheit Robert WBaker Susan MFalk Matthias M<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p>http://www.biomedcentral.com/1471-2121/11/15 |
| spellingShingle | Buckheit Robert W Baker Susan M Falk Matthias M Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes BMC Cell Biology |
| title | Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes |
| title_full | Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes |
| title_fullStr | Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes |
| title_full_unstemmed | Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes |
| title_short | Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes |
| title_sort | green to red photoconvertible fluorescent proteins tracking cell and protein dynamics on standard wide field mercury arc based microscopes |
| url | http://www.biomedcentral.com/1471-2121/11/15 |
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