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 (IMM) potential (a.k.a. ΔΨ m ) that is essential for ATP synthesis, Ca 2+ homeostasis, redox balance and regulation of other key signaling pathways such as mitophagy and apoptosis.However, the detailed mechanisms by which ΔΨ m regulates cellular function remain incompletely understood, partially due to difficulty of manipulating ΔΨ m with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a nextgeneration optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous Channelrhodopsin-2 (ChR2) fusion protein to the IMM and formation of functional cationic channels capable of light-induced selective ΔΨ m depolarization and mitochondrial autophagy. Importantly, we for the first time show that optogenetic-mediated mitochondrial depolarization can be well-controlled to differentially influence the fate of cells expressing mitochondrial ChR2: while 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.

Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance

“…There are several techniques that allow experimental modulation of the PMF, but most are pharmacologic and therefore irreversible and not cell‐ or tissue‐specific. Herein, we take a novel approach to overcome these barriers to precisely control the PMF by using optogenetics, adapting an approach used recently to dissipate the PMF . One family of widely used optogenetic proteins are bacteriorhodopsin‐related light‐activated proton pumps.…”

“…These proteins pump protons across membranes in response to specific wavelengths of light and are often used to study physiology by modulating electrochemical gradients at the plasma membrane . Only recently have precise optogenetic techniques been applied to compartmentalized cellular events using light‐activated proteins targeted to organelles [,, preprint: ,]. 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 to mitochondria and selectively increase the PMF.…”

Mitochondrial light switches: optogenetic approaches to control metabolism

“…Targeting was through N-terminal fusion to 4 repeats of the well-characterized 29 amino acid COX8A MTS (116 amino acids total) [52,58,59]. Our laboratory has found that targeting approaches using a single COX8A targeting sequence are insufficient to direct ChR2 or proton pumps to mitochondria and result instead in plasma membrane targeting, a finding that has been corroborated by others [52,60]. Mitochondrial targeting using the COX8A repeated sequence was achieved only after deleting a small leading section of the ChR2 sequence (24 amino acids) thought to contain a plasma membrane targeting sequence [52].…”

“…Another approach to selectively dissipate the PMF again targeted ChR2 to mitochondria using a different strategy. Similar to initial targeting strategies for mitoChR2 [52], ChR2 did not reach mitochondria using one, two, or three repeats of the COX8A MTS [60]. ABCB-ChR2 was successfully targeted using the large transmembrane ABCB10 MTS [65].…”

“…2A,B); however, orientation in the membrane should not alter the effect of ChR2 as the channel has no directionality. Indeed, the activation of ABCB-ChR2 also resulted in PMF dissipation in cells [60].…”