Tags

Type your tag names separated by a space and hit enter

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes.
BMC Cell Biol 2010; 11:15BC

Abstract

BACKGROUND

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.

RESULTS

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.

CONCLUSIONS

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 (alpha-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.

Authors+Show Affiliations

Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

20175925

Citation

Baker, Susan M., et al. "Green-to-red Photoconvertible Fluorescent Proteins: Tracking Cell and Protein Dynamics On Standard Wide-field Mercury Arc-based Microscopes." BMC Cell Biology, vol. 11, 2010, p. 15.
Baker SM, Buckheit RW, Falk MM. Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes. BMC Cell Biol. 2010;11:15.
Baker, S. M., Buckheit, R. W., & Falk, M. M. (2010). Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes. BMC Cell Biology, 11, p. 15. doi:10.1186/1471-2121-11-15.
Baker SM, Buckheit RW, Falk MM. Green-to-red Photoconvertible Fluorescent Proteins: Tracking Cell and Protein Dynamics On Standard Wide-field Mercury Arc-based Microscopes. BMC Cell Biol. 2010 Feb 22;11:15. PubMed PMID: 20175925.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes. AU - Baker,Susan M, AU - Buckheit,Robert W,3rd AU - Falk,Matthias M, Y1 - 2010/02/22/ PY - 2009/11/21/received PY - 2010/02/22/accepted PY - 2010/2/24/entrez PY - 2010/2/24/pubmed PY - 2010/4/29/medline SP - 15 EP - 15 JF - BMC cell biology JO - BMC Cell Biol. VL - 11 N2 - BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 (alpha-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. SN - 1471-2121 UR - https://www.unboundmedicine.com/medline/citation/20175925/Green_to_red_photoconvertible_fluorescent_proteins:_tracking_cell_and_protein_dynamics_on_standard_wide_field_mercury_arc_based_microscopes_ L2 - https://bmccellbiol.biomedcentral.com/articles/10.1186/1471-2121-11-15 DB - PRIME DP - Unbound Medicine ER -