Optogenetic Control of Lysosome Function

Lysosome protective, metabolic, signaling functions are highly dependent on their pH. A lack of tools of high spatial and temporal resolution for pH control is a bottleneck of lysosome related cell research. Light-driven inward proton pump Ns XeR, targeted to the lysosomes of mammalian cells, produces lysosome alkalization simply by light. Complementary use of outward proton pumping Arch3 rhodopsins in lysosomes offers an approach to vary pH in a range from around 5 to 6.5 in both directions (de-acidification and acidification). Lyso- Ns XeR optogenetics efficiency was demonstrated, in particular, by its ability to inhibit lysosome proteolytic enzymes. Thus, optogenetic monitoring and regulation of the lysosome function, through pH control over a wide range, could serve as an approach to studying fundamental cell processes, and rational drug design.### Competing Interest StatementThe authors have declared no competing interest.* ### Abbreviations Arch3 : archaerhodopsin from Halorubrum sodomense Ns XeR : xenorhodopsin from the nanohalosarchaeon Nanosalina vATPase : Vacuolar-type H+-ATPase, enzyme responsible for maintaining lysosomal pH by ATP-dependent proton pumping TFEB : transcription factor EB ER : endoplasmatic reticulum HEK293T : Human Embryonic Kidney 293 with expression of mutant version of the SV40 large T antigen cell line for immortalization through p53 inhibition BafA1 : Bafilomycin A1, vATPase inhibitor pCMV : expression vector with Cytomegalovirus promoter CD63 : Cluster of Differentiation 63 βHK : β-subunit of H+,K+-ATPase Rab7 : Rab7 protein, marker for late endosomes/lysosomes EEA1 : Early Endosome Antigen 1, marker for early endosomes LTR : LysoTracker Red DND-99, marker of acidic orgenelles ATR : all-trans retinal lyso- : lysosomal SD : standard deviation ROI : region of interest LED : light emitting diode GFP : green fluorescent protein se-pHluorin : super-ecliptic pH-sensitive GFP mutant pHluorin RFP : red fluorescent protein CLSM : confocal light scanning microscope CTSB : cathepsin B FCCP : Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone CCCP : Carbonyl cyanide 3-chlorophenylhydrazone ATP : adenosine triphosphate SH-SY5Y : human neuroblastoma cell line PANC-1 : epithelioid carcinoma attached cell line that is currently used as an in vitro model to study pancreatic ductal adenocarcinoma carcinogenesis and tumour therapies HeLa : cervical carcinoma cell line DMEM : Dulbecco’s modified Eagle’s medium.