Bright Monomeric Photoactivatable Red Fluorescent Protein for Two-Color Super-Resolution sptPALM of Live Cells

详细信息    查看全文

• 作者：Fedor V. Subach ; George H. Patterson ; Malte Renz ; Jennifer Lippincott-Schwartz ; Vladislav V. Verkhusha
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：May 12, 2010
• 年：2010
• 卷：132
• 期：18
• 页码：6481-6491
• 全文大小：669K
• 年卷期：v.132,no.18(May 12, 2010)
• ISSN：1520-5126

文摘

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, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.