Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance.

Brandon J Berry,Yunki Lim,Andrea M Amitrano,Andrew P Wojtovich,Alexander S Milliken,Adam J Trewin,Aksana Baldzizhar,Minsoo Kim

doi:10.15252/embr.201949113

Abstract

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria-targeted light-activated proton pump that we name mitochondria-ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose-dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy-sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well-characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.

Highlights

Mitochondria generate an electrochemical proton gradient known as the protonmotive force (PMF)
PMF is generated by proton pumping respiratory complexes of the electron transport chain (ETC) located in the mitochondrial inner membrane (IM)
By probing the evolutionarily conserved hypoxia adaptation response (Pena, Sherman et al, 2016, Wang, Lim et al, 2019, Wojtovich, Nadtochiy et al, 2012b, Wojtovich, Nadtochiy et al, 2013), we show that tools like mtON allow precise determination of cause and effect in physiologic models

Summary

INTRODUCTION

Mitochondria generate an electrochemical proton gradient known as the protonmotive force (PMF). Rather than using a non-specific cation channel to permeabilize the IM and dissipate the PMF, here we target the light-activated proton pump from the fungal organism Leptosphaeria maculans (Chow et al, 2010, Waschuk, Bezerra et al, 2005) to mitochondria and selectively increase the PMF. We call this optogenetic tool mitochondria-ON (mtON) due to its ability to mimic the proton pumping activity of the ETC in response to light, independent of oxygen or substrate availability. By probing the evolutionarily conserved hypoxia adaptation response (Pena, Sherman et al, 2016, Wang, Lim et al, 2019, Wojtovich, Nadtochiy et al, 2012b, Wojtovich, Nadtochiy et al, 2013), we show that tools like mtON allow precise determination of cause and effect in physiologic models

RESULTS & DISCUSSION

B Confocal images demonstrate overlap of GFP tagged mtON with MitoTracker

MATERIALS & METHODS