However, these techniques are limited in that photobleached protein populations may not be tracked beyond the point of photobleaching. Protein kinetics and rates of protein exchange are typically determined through the use of techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence loss in photobleaching (FLIP). In the dark, these proteins mature to a green fluorescent state with a half-time of about 90 minutes at 37☌, while irradiation with UV-violet light (and also intense blue light in case of Dendra2) results in their irreversible transition into a very photostable, bright-red fluorescent state that can be tracked for hours and days without significant photobleaching on a fluorescence microscope. Monomeric Anthozoa-derived green-to-red photoconvertible fluorescent proteins (Dendra2, mEos2, mKikGR) showed particular promise for improved methods of tracking the dynamics of discrete protein pools within cells. A few years ago, encouraged by the successful use of GFP and of other fluorescent proteins, several laboratories began to develop photoactivatable and photoconvertible fluorescent proteins (PAFPs), which typically undergo a pronounced increase or shift in their spectral emission properties in response to UV-violet (350-420-nm) or, in case of Dendra2 also intense blue light (488 nm) 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.Įxpression of GFP-fusion proteins in live cells revolutionized cell biology by allowing for visualization and tracking of proteins of interest in real time at high spatio-temporal resolution. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. 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. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. 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. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |