Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

Hiromi Imamura,Akira Kakizuka,Silvia Penuela,Shin Mizukami,Dale W Laird,Kazuya Kikuchi,Shuichiro Sakamoto,Tomoki Yoshida,Yusuke Matsui

doi:10.7554/elife.61960

Hiromi Imamura, Akira Kakizuka + Show 7 more

Open Access

https://doi.org/10.7554/elife.61960

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Abstract

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Highlights

Living cells require energy that is provided by the principal intracellular energy carrier, adenosine-triphosphate (ATP)
To reveal the dynamics of intracellular ATP levels during apoptosis at the single-cell level, we first used a genetically encoded Förster resonance energy transfer (FRET)-based ATP biosensor, ATeam (Imamura et al, 2009), which is comprised of a cyan fluorescent protein (CFP; mseCFP), an FoF1-ATP synthase e subunit and yellow fluorescent protein (YFP; cp173-mVenus)
O-DEVD-FR, which enabled dual imaging of both ATP levels and caspase-3 activities during the apoptotic process at the single-cell level. This method allowed us to analyze the single-cell dynamics of cytosolic ATP level after caspase-3 activation, which occurs at different times in different cells

Summary

Introduction

Living cells require energy that is provided by the principal intracellular energy carrier, adenosine-triphosphate (ATP). Apoptosis is a form of programmed cell death, with important roles in development, tissue homeostasis, and immunity. It is characterized by distinctive morphological changes, such as membrane blebbing, nuclear condensation, and externalization of phosphatidylserine (Elmore, 2007). Because cleavage of specific target proteins by activated effector caspases triggers apoptotic events, including the characteristic morphological changes, apoptotic cell death is a systematically and genetically determined, or ‘programmed’, process. Considered to be an energy-demanding process, requiring intracellular ATP for the execution of the cell death program. The molecular mechanism of how intracellular ATP decreases in apoptotic cells remains to be elucidated. We found that the intracellular ATP level starts to decrease following the activation of caspase-3 and that the caspase-triggered opening of the plasma membrane channel pannexin-1 (PANX1) is the major cause of the decrease in intracellular

Results

Discussion

Materials and methods