Baker Susan M, Buckheit Robert W, Falk Matthias M
Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.
BMC Cell Biol. 2010 Feb 22;11:15. doi: 10.1186/1471-2121-11-15.
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.
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.
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.
绿色荧光蛋白（GFP）及其他荧光蛋白融合物已被广泛用于追踪活细胞中的蛋白质动态。最近，光激活、光开关和光转换荧光蛋白（PAFP）的发展使得研究标记蛋白离散亚群的命运成为可能。当克隆/改造单体变体（如Dendra、mEos和mKikGR）时，克服了其最初使用时的局限性（由于其四聚体性质）。
在此，我们报告，通过关闭视场光阑，在配备标准DAPI、荧光素和罗丹明滤光片组以及汞弧照明的宽场荧光显微镜上，在5 - 10秒内实现了离散亚细胞蛋白池的选择性、精确且不可逆的绿色到红色光转换（330 - 380 nm照明）。使用带有长通发射滤光片（LP420）的DAPI滤光立方体可实时观察和控制光转换过程。光转换后，对活细胞成像长达5小时，通常未检测到光毒性或光漂白现象。
我们以Dendra2和mEos2作为单体、光转换PAFP代表，与具有低（组蛋白H2B）、中（间隙连接通道蛋白连接蛋白43）和高（α - 微管蛋白；网格蛋白轻链）动态细胞迁移率的蛋白质融合为例，证明了该技术的实用性。实现了细胞核、间隙连接、微管和网格蛋白包被小泡选定部分的高效、不可逆的绿色到红色光转换。随时间追踪可阐明这些亚细胞结构的动态生命周期。该技术的优点是可以在配备汞弧照明的标准、相对廉价的宽场荧光显微镜上进行。与先前描述的基于激光扫描共聚焦显微镜的光转换方法一起，该技术有望进一步提高光转换PAFP在追踪细胞和蛋白质随时间动态运动方面的通用性。
