iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP

• 成果类型: Article
• 署名作者: Marvin, Jonathan S.; Kokotos, Alexandros C.; Kumar, Mukesh; Pulido, Camila; Tkachuk, Ariana N.; Yao, Jocelyn Shuxin; Brown, Timothy A.; Ryan, Timothy A.
• 署名单位: Howard Hughes Medical Institute; Cornell University; Weill Cornell Medicine; Johns Hopkins University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10259
• DOI: 10.1073/pnas.2314604121
• 发表日期: 2024-05-21
• 关键词: mitochondrial; fluctuation; cells

摘要

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real - time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP - binding helices of the epsilon - subunit of a bacterial F-0 - F-1 ATPase. Optimizing the linkers joining the two domains resulted in a similar to fivefold to sixfold improvement in the dynamic range compared to the previous - generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 mu M to 500 mu M. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single - synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.