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Optogenetic tools could be even more powerful if THE AUTHOR FILE two proteins were expressed in the same ratio in one cell—one would activate a neuron, and another would silence it. Such control could allow incredibly precise Ernst Bamberg experiments about what happens when certain neu- Fusing light-activated proteins for precise rons fire as well as when they are prevented from firing. optogenetic control In practice, such experiments have been very chal- lenging. When cells are made to express two optoge- Ernst Bamberg has grown used to surprises from netic proteins, they tend to make too many copies of microbial rhodopsins. About a decade ago, he, togeth- one and too few of another, and ratios vary from cell er with Georg Nagel and Peter Hegemann, character- to cell. Even when genes for both proteins are placed ized the proteins that on the same construct, differences in how proteins are help green algae move processed make it impossible to control how many toward light. Unlike functional copies of the hyperpolarizing molecules previously known exist for every depolarizer. light-sensing proteins, To solve this problem, Bamberg and colleagues such as light-activated attempted an apparently simple solution: they created bacterial ion pumps, a fusion protein in which the two optogenetic tools algal rhodopsins are remained physically attached. Not only would they light-gated ion chan- be expressed together, they would be placed into the nels that operate on a plasma membrane together. much faster time scale. The details were harder to deal with. If channel-

P. Lastrico, MPI Biophysics Lastrico, P. Scientists had trou- rhodopsin and halorhodopsin were physically linked Ernst Bamberg ble believing that a together directly, one rhodopsin would turn the other rhodopsin-like mol- upside down. “You need another helix in between,” ecule can act as a cation channel. “We had a hard time explains Bamberg. To link the proteins, Bamberg chose trying to convince people that it was true,” Bamberg a section of a rat gastric recalls. However, Bamberg says he’d already realized proton ATPase as well as a “This is still the potential of these light-gated channels, or chan- fluorescent protein. for me a little nelrhodopsins. “Before we published the first papers The very first construct [showing that algal proteins could generate currents looked good, at least ini- miracle” in eukaryotic cells], we applied for a patent where we tially. The fusion construct gave to our fantasy a free run about the possible appli- seemed to be appearing at the cell membrane. “We saw cations of channelrhodopsins on electrically excitable something and we were happy,” says Bamberg. “Then Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 cells, including some biomedical applications.” (The we analyzed the data and we saw that it didn’t work patent was recently licensed by a large pharmaceutical properly.” Setbacks are to be expected, says Bamberg.

© 201 company for a project on gene therapy to restore vision “Science is such. You cannot make a plan and say this in the eye.) has to work.” The scientific community is now convinced: chan- They kept at it: changing the length of the linker, nelrhodopsins have become a central part of a new switching which side of the linker each rhodopsin was technology called optogenetics. Researchers across the on. Finally, after creating some two dozen constructs, world have reported using channelrhodopsins to con- the researchers obtained convincing results. The paired trol electrical signals in neurons as well as cardiomyo- proteins could be used to precisely control membrane cytes, sometimes within living mice and other animals. potential in cultured neurons. These applications came as yet another surprise, says As a bonus, this linker promises to work for sev- Bamberg. Few expected these microbial membrane eral optogenetic tools. In addition to combining proteins to function in animal cells. “This is still for channelrhodopsin-2 and halorhodopsin, the me a little miracle,” says Bamberg, at the Max-Planck- researchers were able to join the blue light–absorbing Institut für Biophysik in Frankfurt. channelrhodopsin-2 with the yellow light–­absorbing In fact, researchers who want to use light to control channelrhodopsin-1, allowing the activation of processes in animal cells now have several kingdom- nerve cells over a wide range of the spectrum of vis- crossing proteins to choose from. The proteins even ible light. “Now we have a cassette where you can perform different tasks. Stimulated by blue light, put all kinds of rhodopsins together,” says Bamberg. channelrhodopsin-2 allows cations to move across Monya Baker the cell membrane, depolarizing cells and caus- Kleinlogel, S. et al. A gene-fusion strategy for ing neurons to fire. Stimulated by orange light, stoichiometric and co-localized expression of halorhodopsin pumps chloride ions across the cell light-gated membrane proteins. Nat. Methods 8, membrane, causing hyperpolarization and prevent- 1083-1088 (2011). ing neurons from firing.

nature methods | VOL.8 NO.12 | DECEMBER 2011 | 985