Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
复制标题
DOI：
10.1021/ja100906g
复制
发表时间：
2010-05-12
影响因子：
15
通讯作者：
Verkhusha VV
中科院分区：
文献类型：
--
作者：
Subach FV;Patterson GH;Renz M;Lippincott-Schwartz J;Verkhusha VV
快速出现的活细胞超分辨率单分子显微镜技术依赖于遗传编码的光活化荧光蛋白的持续发展。在单体TagRFP的基础上，我们开发了一种可光活化的TagRFP蛋白，该蛋白最初是暗的，但在紫光照射后变成红色荧光。与包括PAmCherry在内的其他单体深红色光活化蛋白相比，PATagRFP具有显著更高的分子亮度、更好的pH稳定性、显著更低的蓝光敏感性以及在整体和单分子模式下更好的光稳定性。光谱分析表明，PATagRFP光活化是一个两步的光化学过程，涉及两个不同的发色团形式的顺序单光子吸收。真正的单体行为，没有绿色荧光，和活细胞中的单分子性能，使PATagRFP的双色成像技术，包括传统的衍射限制光活化显微镜，超分辨率光活化定位显微镜（PALM），和单粒子跟踪PALM（sptPALM）的活细胞的一个优秀的蛋白质标签。使用几种PATagRFP标记的跨膜蛋白以及PAGFP标记的网格蛋白轻链证明了双色sptPALM成像。对所得sptPALM图像的分析显示，通过内吞作用内化到细胞中的单分子跨膜蛋白在空间和时间上与富含网格蛋白轻链分子的质膜结构域共定位。
Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP tagged clathrin light-chain. Analysis of the resulting sptPALM images revealed that single molecule transmembrane proteins, which are internalized into a cell via endocytosis, co-localize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.
登录
查看更多内容
DOI：
10.1073/pnas.97.22.11984
发表时间：
2000-10-24
影响因子：
11.1
作者：
Baird, GS;Zacharias, DA;Tsien, RY
通讯作者：
Tsien, RY
影响因子：
46.9
作者：
Chudakov, DM;Belousov, VV;Lukyanov, KA
通讯作者：
Lukyanov, KA
影响因子：
56.9
作者：
Patterson, GH;Lippincott-Schwartz, J
通讯作者：
Lippincott-Schwartz, J
影响因子：
48
作者：
Manley, Suliana;Gillette, Jennifer M.;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer
DOI：
10.1073/pnas.0700059104
发表时间：
2007-04-17
影响因子：
11.1
作者：
Henderson, J. Nathan;Ai, Hui-wang;Remington, S. James
通讯作者：
Remington, S. James