Precisely Control Mitochondria with Light to Manipulate Cell Fate Decision

Abstract

Mitochondrial dysfunction has been implicated in many pathological conditions and diseases. The normal functioning of mitochondria relies on maintaining the inner mitochondrial membrane potential (also known as DJm) that is essential for ATP synthesis, Ca 2þ homeostasis, redox balance, and regulation of other key signaling pathways such as mitophagy and apoptosis. However, the detailed mechanisms by which DJm regulates cellular function remain incompletely understood, partially because of the difficulty of manipulating DJm with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a next generation optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous channelrhodopsin-2 fusion protein to the inner mitochondrial membrane and formation of functional cationic channels capable of light-induced selective DJm depolarization and mitochondrial autophagy. Importantly, we for the first time, to our knowledge, show that optogenetic-mediated mitochondrial depolarization can be well controlled to differentially influence the fate of cells expressing mitochondrial channelrhodopsin-2; whereas sustained moderate light illumination induces substantial apoptotic cell death, transient mild light illumination elicits cytoprotection via mitochondrial preconditioning. Finally, we show that Parkin overexpression exacerbates, instead of ameliorating, mitochondrial depolarization-mediated cell death in HeLa cells. In summary, we provide evidence that the described mitochondrial-targeted optogenetics may have a broad application for studying the role of mitochondria in regulating cell function and fate decision.

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

“…Using the mitochondria‐ON (mtON) construct we determined the temporal effect by the acute reversibility of light exposure. Our result was supported by evidence in cells using a similar optogenetic approach to spatiotemporally control the PMF, showing brief PMF loss preconditions cells to be resistant to later, severe PMF disruption 23 . Decreasing the PMF during hypoxia to relieve oxidative stress is thoroughly characterized 24–28 ; here we address how decreasing the PMF prophylactically signals protection against impending hypoxic insults in vivo through energy sensing.…”

“…Our construct was oriented to pump protons from the mitochondrial intermembrane space (IMS) into the matrix to dissipate the PMF (Figure 1A). We called this construct mitochondria‐OFF, or mtOFF, due to its ability to “turn off” mitochondrial function through the PMF in response to light, as validated here and by other studies using ChR2 23,48 . The mitochondrial targeting sequence (MTS) and part of the coding sequence of the SDHC1 protein were used to direct and orient mitochondrial expression of Mac.…”

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance

“…Using the mitochondria-ON (mtON) 87 construct we determined the temporal effect by using the acute reversibility of light exposure. Our result was 88 supported by evidence in cells using a similar optogenetic approach to spatiotemporally control the PMF, 89 showing brief PMF loss preconditions cells to be resistant to later, severe PMF disruption (Ernst et al, 2019).…”

Lights up on organelles: Optogenetic tools to control subcellular structure and organization

“…These strategies have been applied to questions of fundamental biology and have increasingly benefited biotechnological endeavors. Monomeric channelrhodopsins have elucidated the interplay between mitochondrial membrane potential and cellular calcium handling (Ernst et al, 2019;Tkatch et al, 2017) and photocaging of nuclear translocation signals has allowed for optogenetic regulation of cell division (Wehler et al, 2016). Dimeric systems have enabled biologists to interrogate both site specific enzymatic activity (O'Banion et al, 2018;Valon et al, 2017) and the effect of subcellular location upon organelle function (Griesche et al, 2019).…”

“…ChRs, which have historically been used to modulate the activity of neurons optogenetically, can also be employed to alter mitochondrial membrane potential. Light activation of mitochondrially targeted ChRs leads to disruption of the electrochemical gradient, alters mitochondrial calcium handling, and impacts cell viability (Ernst et al, 2019; Tkatch et al, 2017) (Figure 5a). While light‐induced mitochondrial membrane depolarization has been shown to initiate autophagy, optogenetics also provides avenues to directly induce cell death through localization of apoptosis regulators such as Bcl‐2‐associated X protein (BAX) (Hughes et al, 2015).…”