Mammalian cells calcium imaging They performed calcium imaging experiments on HEK293T cells. They regularly tested the cells for mycoplasma contamination using the MycoAlert kit and confirmed they were negative. They seeded the cells in 35-mm culture plates and transfected them at 70-80% confluency using jetOPTIMUS in a 1:1 ratio with a total of 1 μg of DNA. After one day of expression, they bathed the cells in standard bath solution and performed video imaging using Micro-Manager 1.4.21 software on a Zeiss Axiovert 200M epifluorescence microscope. They used a Thorlabs M470L4 LED, a Zeiss Filter set 44, a Zeiss LD Achroplan 40X/0.6 Korr 440864 objective, and an Andor Zyla sCMOS camera. They acquired images at 10 fps and extracted fluorescent values from individual cells using ImageJ 1.54d software. They also obtained confocal images from cells seeded in 25-mm poly-L-lysine-treated coverslips using MetaMorph 7.10.5.476 software on a Nikon TiE microscope. They used a 40X objective with Ilas2 illumination module set to 488 nm, a Yokogawa CSU W1 confocal scanner unit, and an Andor iXon Life 888 (emCCD) camera. In some cases, they incubated the cells for 2 hours with 15 μM BAPTA-AM in DMEM supplemented with 10% FBS at 37 °C and then bathed them in standard bath solution with the same concentration of BAPTA-AM during imaging.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.