Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

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.

Figure 1

Gap junction (GJ) dynamics revealed by photoconverting expressed Cx43-Dendra2. (A) GJs consisting of many densely packed channels visible as green lines and puncta between HeLa cells expressing Cx43-Dendra2 (panel 1). GJs orient perpendicular providing a view onto their edge (circled in center, panel 1; also shown in Figures 1B, C), or horizontally providing a view onto their surface (circled on right; remaining panels, Figure 1A). GJs are dynamic structures. Their channels are replaced within several hours, as demonstrated by photoconverting Dendra2-tagged Cx43. A region containing two horizontally oriented GJs was photoconverted (entire field shown), and green and red channels were recorded over time. Within 1-hour post conversion a widening, homogenous green line of channels appeared along the GJs (panels 2-5). (B) Following photoconverted GJs for longer periods resulted in a steady loss of red fluorescence from the photoconverted area, and a simultaneous recovery of green fluorescence (circled in panel 1), suggesting that older channels are continuously removed from central GJ areas, while newly synthesized channels are simultaneously added to their periphery. Fluorescence intensity profiles for red and green channels measured along lines traversing the photoconverted GJs are shown. (C) Photoconversion allows estimation of GJ channel turnover. A portion of a perpendicular oriented Cx43-Dendra2 GJ was photoconverted (circled). Over time, red fluorescent puncta appeared adjacent to the converted GJ area (arrow-heads, panels 3, 4). Puncta were not detected immediately post-conversion (panel 2), suggesting that they were released from the photoconverted GJ area. Puncta correspond to degradative endocytic vesicles that are generated by the release of small GJ channel packets from GJs [15]. (D) Schematic representation of GJ turnover as shown experimentally in (A-C).

Figure 2

Histone 'dynamics' revealed by photoconverting expressed Dendra2-H2B. Entire cell nuclei (in A, and top row in B), or portions of nuclei (center and bottom rows in B) of Dendra2-H2B expressing HeLa cells were photoconverted within 5-10 sec (circled areas), and green and red channels were imaged immediately after photoconversion (in A), and after 1-hour (in B). One-hour post conversion, the areas of photoconverted histone H2B protein and the edges between photoconverted and unconverted Dendra2-H2B domains were still well defined, revealing H2B's stable association with DNA in interphase chromatin. DIC and fluorescence images were merged in (A) to reveal the location of cell nuclei, and transiently transfected Dendra2-H2B expressing cells. Note that more or less efficient photoconversion of cell nuclei was achieved (compare remaining green fluorescence in the circled areas in B, rows 1 and 3, compared with row 2).

Figure 3

Microtubule dynamics revealed by photoconverting expressed Dendra2-α-tubulin. A distal portion of a Dendra2-α-tubulin expressing HeLa cell was photoconverted within 5 sec (entire field shown in the panels). Note, that photoconversion was only incomplete, as indicated by the relatively strong remaining green fluorescence (visible in the left panel of row 2). Within 3-7 minutes, red photoconverted and remaining green α-tubulin pools intermixed (arrows), consistent with the known dynamic continuous polimerization and depolimerization of microtubules, and the diffusional mobility of unassembled α/β-tubulin subunit-dimers in the cytoplasm.

Figure 4

Clathrin-coated vesicle dynamics revealed by photoconverting expressed mEos2-clathrin light chain. Numerous clathrin-coated vesicles are visible in the cytoplasm of an mEos2-clathrin light chain expressing HeLa cell (panel 1, row 1). A distal portion of this vesicle pool was photoconverted within about 10 sec (circled). Within 2-5 minutes after photoconversion, photoconverted and remaining, unconverted vesicles moved laterally and intermixed (red, green and yellow vesicles, the resulting color when red and green fluorescence colocalize, arrows), consistent with the known dynamic mobility and structural composition of these vesicles. N = Cell nucleus.