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Home , Channelrhodopsin, Halobacterium salinarum

Halobacteria belong to the archaebacteria. They have a particular preference for extremely saline environments. FOCUS_Optogenetics

Single-Celled Organisms Shed Light on Neurobiology

The discovery of a visual pigment in the cell membrane of an archaebacterium in the early 1970s is owed solely to a researcher’s curiosity: For three years, the scientific community wouldn’t believe Dieter Oesterhelt. Forty years after his pioneering work at the Max Planck Institute of Biochemistry in Martinsried, and channelrhodopsin, which stems from a single-celled green alga, are gaining ground as new tools in neurobiology.

TEXT CHRISTINA BECK

t was an illustrious group that the to revolutionize the neurosciences, per- It was more or less a coincidence that Royal Swedish Academy of Sciences mitting, as it does, the first ever non-in- brought biochemist Dieter Oesterhelt invited to the Lilla Frescativägen in vasive manipulation of neural networks into contact with salina- Stockholm on December 12, 2013. in an organism – from the small nem- rum. But this archaebacterium would was the topic on which atode Caenorhabditis elegans to the eventually become the central research I the Nobel Prize Committee was seeking mouse. Someday, perhaps, even in hu- object of his scientific life for the next information. Among the eleven scien- mans. In other words, a method that 40 years. Oesterhelt had done his doc- tists were two from Max Planck insti- has the potential to win a Nobel Prize. torate in Feodor Lynen’s lab at the Max tutes, as well as two other researchers Planck Institute for Cell Chemistry (lat- who had taken their first steps in this THE DISCOVERY OF THE FIRST er the Max Planck Institute of Biochem- field as young group leaders at Max MICROBIAL istry) on a metabolic enzyme, fatty acid Planck. synthase. “A giant particle,” as he says, As early as 1979, the discoverer of But what has since become a popular “whose structure could be decoded only the DNA code, Francis A. Crick, called tool of neuroscientists had its begin- through electron microscopy.” it the greatest challenge in the neuro- nings somewhere else entirely: in a small, This explains why the researcher sciences: to selectively influence a spe- halophilic archaebacterium, Halobacte- went on a sabbatical in 1969, to San cific cell type in the brain and leave rium salinarum. Archaebacteria are the Francisco and Walther Stoeckenius, a the others unchanged. In his lectures, “old-timers” of life. Since the early days renowned expert in the field of elec- he speculated that light could serve as of evolution, these single-celled organ- tron microscopy. Oesterhelt wanted to a control tool – in the form of locally isms have persevered in extreme habi- learn to use this technology in the lab and temporally restricted impulses of tats – in lakes, for instance, or hot with him. different colors. volcanic springs – while and Stoeckenius was interested in the Thirty years later, this vision is be- the eukaryotes were able to develop membrane of the halobacterium be-

Photo: POWER AND SYRED/SPL/Agentur Focus coming a reality: optogenetics is poised much more freely. cause, at that time, the molecular struc-

4 | 14 MaxPlanckResearch 19 FOCUS_Optogenetics

1969

After completing his doctorate under Feodor Lynen (right) at the Ludwig-Maximilians-Universität Institute of Biochemistry, and a two-year postdoc phase at the Max Planck Institute for Cell Chemistry, young biochemist Dieter Oesterhelt (left) went on a sabbatical to work with Walther Stoeckenius at the University of California, San Francisco in 1969. There he surprisingly succeeded in detecting a rhodopsin-like (bacteriorhodopsin) in the membrane of Halobacterium salinarum.

ture of cell membranes was still a sub- membrane using mass spectroscopy. equipment. The Max Planck Institute ject of controversy. “That was the so- No doubt about it: it was . Wal- for Cell Chemistry moved out to Mar- called purple membrane, which is how ther Stoeckenius’ initial reaction, how- tinsried, while Oesterhelt remained in it was known even then. But it was ever, was less than euphoric – he said, the originally shared labs at the Lud- completely unclear what it was,” says quite simply: “That can’t be. That wig-Maximilians-Universität Institute Dieter Oesterhelt. doesn’t exist in prokaryotes.” of Biochemistry. “All I had left was a pH Allen Blaurock, who was likewise in The reviewer for the journal NATURE meter, a water bath and a projector,” he Stoeckenius’ lab at the time, requested likely had a similar view. The submit- says. But this situation proved to be a Oesterhelt’s assistance in preparing his ted publication was rejected with a blessing for the key experiment that samples. Oesterhelt experimented with note saying that the experiments were followed, “because I simply couldn’t do different organic solvents to elute the fine, but the analogy with rhodopsin much more.” lipids from the membrane: “So I ex- was pretty far-fetched. “It was simply Oesterhelt was firmly convinced that tracted the purple membrane with unacceptable to find retinal some- the color change is associated with a chloroform/methanol – and suddenly I where other than in an eye,” con- function, so he worked on reversing it: had a yellow extract,” he recalls. cludes Oesterhelt. So the first publica- “Quite simply, I tried every solvent in Such a change in absorption across tion on bacteriorhodopsin, as the the world.” And then here, too, coinci- an area of nearly 200 nanometers authors had christened their molecule, dence again played a role. Specifically, if seemed quite unusual to the young appeared in 1971 in the journal NATURE I took ether, added salt, and then went biochemist. But Allen Blaurock dis- NEW BIOLOGY. to the window when the Sun was shin- missed it – he had worked on frog ret- ing, the extract suddenly turned bright inas with Maurice Wilkins in London. INVENTED yellow; in the dark, the color changed Their X-ray experiments required NOT ONCE, BUT TWICE back. That was the desired color change, them to irradiate the frog retina at a but what was behind it? very specific angle. “But if we weren’t Dieter Oesterhelt returned to Munich “I simply placed a pH electrode in careful,” Blaurock told Oesterhelt, “the and – despite serious doubts on the part it,” says Oesterhelt. When the color beam went into the frog’s lovely red of his colleagues there – continued to changed from purple to yellow, protons eye, and suddenly it turned yellow.” work with the bacteriorhodopsin: “It were released, and when the color That was the decisive clue for Diet- seems to me to be quite an unusual changed from yellow to purple, protons er Oesterhelt. He went to the library to thing, and it isn’t there without rea- were taken up. Accordingly, the extract retrieve the data on retinal, the light- son,” he explained to the skeptics. And became acidic in the one case, and al- absorbing pigment in the retina of ver- then the shortage of collaborators was kaline in the other. However, when

tebrates, and then analyzed the purple also accompanied by a shortage of such a release and uptake of protons Photo: MPG/Krella

20 MaxPlanckResearch 4 | 14 Photo: MPG/Wolfgang Filser; graphic: Wikimedia/Darekk2/Creative Commons Attribution Share-Alike 3.0 interior ofthebacteriacell, aproton deed, alight-drivenprotonpump. proof thatbacteriorhodopsin is,in- vant readingsand,with them, the a fewdays,hehadgathered therele- dle shotstraighttotheupperlimit.”In “The recordergaveajerkandthenee- sitivity andthenturnedthelighton: He setthepHmetertohighestsen- happen whenheexposedintactcells. ter inthedarkroomtoseewhatwould suspension ofbacteriacells. possible tomeasureapHchangein cule pumpsprotons,thenitshouldbe ly hopethatyou’reright.”Ifthemole- simply: “Idon’t believeit,butIcertain- FeodorLynen.tion supervisor Hesaid helt presentedthisideatohisdisserta- and releasesthemintheother. Oester- takes upprotonsfromonedirection proton pump–thatis,amolecule pumping effect. membrane, itwouldhavetocreatea takes placeinadenselayer, suchasa By transportingprotonsout ofthe Dieter OesterheltsetuphispHme- The youngbiochemistimagineda » It wassimplyunacceptabletofindretinal somewhere otherthaninaneye.« the cell. triphosphate), theenergycurrencyof zymatic synthesisofATP (adenosine protons flowingbackinisusedforen- Planck researcher. Theenergyofthe charging abattery,” explainstheMax membrane. “Theprocessisjustlike trical potentialisbuiltupacrossthe tween insideandoutside,anelec- concentration gradientiscreatedbe- twice,” saysDieterOesterhelt. process ofphotosynthesisnot once,but evolution inventedthefundamental ple oflivingnature.“Inother words, second light-energyconversion princi- chlorophyll systemofgreen plants, the ple membranesystemis,nexttothe thus providedinitialevidence.Thepur- 1978 –forwhichbacteriorhodopsin in him theNobelPrizeinChemistry Mitchell backin1961–whichearned ic hypothesisproposedbyPeterD. This wasinlinewiththechemiosmot- MEMBRANE EVEN MORELIGHT-SWITCHED rhodopsin Bacterio- synthase ATP Environment fluid Intracellular Cell membrane ing chlorideionsintothe cell.This out ofthecell,butrather, it waspump- protein wasn’t pumpingsodiumions fornia showedthatthismembrane Schobert fromtheUniversity ofCali- sin. ButthenJanosLanyiand Brigitte pump, whichwascalledhalorhodop- inally supposedtoisolatethissodium chemist PeterHegemann.Hewasorig- his firstdoctoralstudentstherewas inMartinsried,andoneof chemistry tor attheMaxPlanckInstituteofBio- activatable sodiumpump. long speculatedthatthiswasalight- fered frombacteriorhodopsin.Itwas of ther pigmentinthepurplemembrane Yagi andMukohatadiscoveredafur- 1977, Japaneseresearchers Matsuno- publications onthistopiceachyear. In there havebeenmorethanahundred start ofthesecondhalf1970s, ology andstructuralbiology. Sincethe subject inbioenergetics,membranebi- rhodopsin rosetobecomeamodel In theyearsthatfollowed,bacterio- Halobacterium salinarum Oesterhelt hadsincebecomeDirec- 1973 currency ofthecell(right). triphosphate), theenergy ic synthesisof ATP (adenosine ing backinisusedfor enzymat- The energy oftheprotons flow- between insideandoutside. ton concentration gradient the bacteriacell,creating apro- protons outoftheinterior activated bylight,transports membrane, theionpump, purple toyellow (left).Inthe extracts tochangecolorfrom light –causesthemembrane uptake ofphotons–thatis, light-driven proton pump. The that bacteriorhodopsinisa Oesterhelt wasabletoshow years later,A few short 4 | 14 MaxPlanckResearch MaxPlanckResearch , butitdif- 21 conditions areparticularlyfavorable for domonas 22 As aphotosyntheticorganism, APPARATUS WAS PUZZLING AN ALGA’S RED EYESPOT road wasrocky, andextremelylong. readers ofamedicaljournal.”Butthe teria couldonedaybeofinteresttothe light-driven iontransportinarchaebac- ofsingle-celledalgaeorthe on themolecularmechanismsof er, we hadn’t expectedthat theresearch experiments morethanadecadeearli- later wrote:“Whenweconductedour CINE reinhardtii unicellular greenalga self toanewobjectofstudy:thesmall, and, inthelate1980s,dedicatedhim- partment ofmembranebiochemistry a groupofhisowninOesterhelt’s de- significance. would latertakeonanentirelynew MaxPlanckResearch MaxPlanckResearch In 1986,Hegemannbeganleading , heandhisco-authorGeorgNagel » seeksoutareaswherethelight . InEMBOM the readers ofamedicaljournal.« We algaecouldoneday expectedthattheresearch interest hadn’t onsingle-celled 4 | Chlamydomonas 14 OLECULAR

Chlamy- M EDI - 19th century. Thelightsensorrespon- totaxis. They’vebeenknownsincethe trolled orientationmovementsaspho- tions. Scientistsrefertosuchlight-con- be adaptedtochanginglightcondi- green algadoesn’t have tocontinually tosynthesis apparatusofthesmall breaststroker. Thismeansthatthepho- long flagellatopropelitselflikealittle photosynthesis. Indoingso,itusesits ers’ fielddidn’t nal. ments in“blind”algaebyadding reti- in restoringthelight-driven move- more, afewyearslater, he succeeded the lightsensorisarhodopsin. Further- postulated –alreadybackin1980that ing theseso-calledactionspectra,he about thelightsensor’s properties.Us- length inordertoobtaininformation monas phototactic movementsof ga’s redeyespotapparatus. sible forphototaxisislocatedintheal- dent ofMaxDelbrück,studiedthe Kenneth W. Foster, aformerstu- “But thephotoreceptorresearch- asafunctionofthelight’s wave- recognize the Chlamydo- signifi- was quiteobviouslynottransmitted via unlike inthehumaneye, current but theyalsorevealedsomething else: tor did,infact,havetobearhodopsin, not onlyshowedthatthephotorecep- lished inthejournalN ising results:thephotocurrentspub- logical measurementsproducedprom- Chlamydomonas dopsins intheeyespotapparatusof know thattherearetendifferentrho- image,” hesays.Today, theresearchers labels yieldedacompletelyundefined mained unsuccessful:“Theradioactive tein chemicalanalyses,butthetestsre- amounts andappropriate the alga’s photoreceptorinsufficient gemann laboredfortenyearstoobtain terest. Together withhiscolleagues,He- one inthehumaneyearousedhisin- similartothe ment thatmaybevery gle-celled greenalgausesavisualpig- says PeterHegemann. cance ofthesefindingsatthetime,” Back then,onlytheelectrophysio- However, thecluethatasmall,sin- . ATURE purity for in1991 pro-

Photo: David Ausserhofer for DFG Photo: Wolfgang Bettighofer 2010/Creative Commons License V 3.0 (CC BY-NC-SA); graphic: Karl Deisseroth, and Feng Zhang newly decodedgenesequences from search Institutepublishedthousandsof gle channel. brane area,butnotthecurrent ofasin- the ioncurrentsacrossalargemem- ways abletoregisteronlythetotalof pipette. Theresearchers werethusal- vations werecarriedoutusingasuction channel. Allelectrophysiologicalderi- properties ofthisallegedrhodopsin crobial rhodopsin. helt’s ofthefirstmi- previousdiscovery with similarskepticismasforOester- munity counteredtheseexplanations channel itself.”Butthescientificcom- channel complex,orevenformsthe rhodopsin ispartofarhodopsin-ion publication: “We assumethatchlamy- an evenmorepointedstatementina at theUniversityofRegensburg,made had sincebeenappointedtoaposition ter exposure. croseconds (millionthsofasecond)af- extremely quickly, withinjust30mi- channel, asthephotocurrentsappeared peared tobecoupleddirectlyanion plified. Instead,thephotoreceptorap- a chemicalsignalcascadeandthusam- in commonwiththetwoionpumpsbacteriorhodopsinandhalorhodopsin. membrane protein, thefirstexampleofadirectly light-gatedionchannel.Channelrhodopsin(right)hasmany structural feature (image onp.22)intheearly1990sshowed until2002thatitcouldbeproven –arhodopsin.Butitwasn’t thatitisacomplet light. The actuallightsensoris–aselectrophysiological measurements takenbytheteamworkingwithPeterHegemann green alga The small,single-celled 2002 – 1991 2000, theJapaneseKazusa DNARe- A newapproachwasneeded. In It stillwasn’t possibletolistthekey Eight yearslater, Hegemann,who Chlamydomonas reinhardtii crobial .Inordertodocu- trophysiological characterizationofmi- accrued yearsofexperienceintheelec- coded bythesegenesequences. test thepropertiesofproteinsen- ics inErnstBamberg’s department,to based MaxPlanckInstituteforBiophys- a research group leaderattheFrankfurt- terial rhodopsingenes. longer sectionsthatweresimilartobac- searchers inRegensburg discoveredtwo ing throughthesesequences,there- cessible onlinedatabases.Whenlook- Chlamydomonas reinhardtii the firstexampleofadirectly light- that thealgaerhodopsinwas indeed S presented thefindingsin journal monas proteins originatingfrom tested theelectricalpropertiesof Nagel andhiscolleaguesnowlikewise THE BIRTH OFOPTOGENETICS them totheeggcellsofclawfrogs. tions, theresearchers hadtransferred under electricallycontrolledcondi- bacteriorhodopsin andhalorhodopsin ment thetransportcharacteristicsof fluid Intracellular CIENCE Hegemann askedGeorgNagel,then Bamberg’s departmenthadalready infrogeggs.InJune2002, they . Itwasthelong-awaitedproof hasared eyespot apparatus (left)thatallowsittoorientitselfusing Bacteriorhodopsin (BR) H

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+ Lys + infreelyac- Chlamydo- (HR) CI

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+ Lys - weeks later. package fromGermanyarrived afew clone ofchannelrhodopsin-2. The the contextofacollaboration, geta Nagel andaskedwhetherhe could,in Deisseroth wroteane-mail toGeorg brain forsometime.InMarch 2004, cellsintheintact cal activityofnerve possibilities forcontrollingtheelectri- searchers hadalreadybeendiscussing den atStanfordUniversity. Thetwore- est ofKarlDeisserothandEdwardBoy- also inhumankidneycells. only infrogeggs,butforthefirsttime, incorporating channelrhodopsin-2not as sodiumions.Theyhadsucceededin other positivelychargedparticles,such to channelrhodopsin-1,alsochannels rhodopsin-2 (ChR2),which,incontrast light-activated ionchannel,channel- lished theirfindingsonthesecond requires noenergy. bacteriorhodopsin, theiontransport or; incontrasttotheprotonpump, via themembraneintocellinteri- tened their“baby,” channelsprotons (ChR1), asthescientistshadchris- taking uplight,channelrhodopsin-1 pletely novelmembraneprotein.After driven ionchannel,andthusacom- This publicationarousedtheinter- One yearlater, theresearchers pub- Channelrhodopsin Na (ChR)

H N +

+ 4 , K Lys | ely novel FOCUS 14 + , Ca MaxPlanckResearch MaxPlanckResearch s 2+ , H _Optogenetics + 23 >

FOKUS_Optogenetik

1995 – 2005

Georg Nagel already managed to express bacteriorhodopsin in animal cells in 1995, in the egg cells of claw frogs (at the top right of the image, on the computer screen), and in this way, to study its electrophysiologi- cal characteristics under controlled condi- tions. But the neuroscientists showed little interest in this. This changed when Nagel succeeded in transferring channelrhodop- sin-2 to frog eggs. Then several research groups dared to attempt, in 2005, to in- corporate channelrhodopsin-2 in nerve cells – and succeeded – in order to use light to control their electrical activity. Only then did the researchers consider using also bacteriorhodopsin as an optogenetic tool. While nerve cells can be activated by light with channelrhodopsin-2, they can be switched off with bacteriorhodopsin.

Boyden, Deisseroth and Feng Zhang, light-activated are indeed suit- with light to suppress action potentials who had subsequently joined them, able for regulating neural networks in in the cell, so it works like an “off” considered how to proceed. In the intact organisms. That was the actual switch. Three years later, Boyden and months that followed, the researchers start of the new research field of opto- his team at MIT were then able to show optimized their experimental setup. Us- genetics. that also the proton pump bacteriorho- ing a harmless virus as a gene ferry, they So it was now possible to use light dopsin that Dieter Oesterhelt had al- managed to introduce the gene for to activate nerve cells within neural cir- ready discovered in the early 1970s is channelrhodopsin-2 into cultivated cuits. The following year, Deisseroth at- capable of switching off. hippocampus neurons. Via the promot- tended a lecture at the Max Planck In- And so we come full circle after er, a genetic switch, they were able to stitute in Frankfurt. He asked his Ger- nearly half a century of basic research. control which type produces man colleagues about an optogenetic What appeared to be a caprice of nature channelrhodopsin-2. The experiment tool that, conversely, permits nerve – a retinal-binding protein in the mem- worked, “incredibly well, actually,” as cells to be switched off using light. brane of an obscure archaebacterium – Deisseroth writes. “Using simple, harm- Bamberg and Nagel told him about is becoming a paradigm for the inter- less flashes of light, we were able to re- their experiments with bacteriorhodop- play between light and life. Today, liably control, with millisecond preci- sin and halorhodopsin in frog eggs in thousands of researchers are using op- sion, when the -producing nerve the mid-1990s. And they recommend- togenetic methods to analyze how ac- cells triggered action potentials.” ed that he use halorhodopsin from Na- tivity patterns of specific neuron groups Together with the Max Planck re- tronomonas pharaonis. Janos Lanyi had control complex physiological process- searchers in Frankfurt, the three Amer- discovered it in 1999. In contrast to es and behaviors. Pioneering work like icans published the findings in NATURE halorhodopsin from Halobacterium sa- that of Zhuo-Hua Pan at Wayne State NEUROSCIENCE in 2005. This break- linarum, it also works at low chloride University show that it isn’t just neuro- through was virtually in the air – in par- concentrations, like those prevailing, biologists who can use optogenetics as allel, Japanese researchers working with for instance, in the mammalian brain. a tool. Hiromu Yawo succeeded in expressing At Stanford, Zhang now synthesized Pan successfully introduced chan- channelrhodopsin-2 in PC12 cells. And the corresponding gene and introduced nelrhodopsin-2 into the retinal cells of Stefan Herlitze (then at Case Western it into nerve cells; at the same time, Al- blind mice, giving them the ability to Reserve University in Cleveland) was exander Gottschalk tested it successful- perceive light again. Other researchers even successful in expressing channel- ly in C. elegans. In spring 2007, the re- have since expanded this approach – re- rhodopsin-2 in the spinal cord of verte- searchers from Frankfurt and Stanford storing eyesight in cases of degenera- brates. He was able to show – just like published their findings: while chan- tive retinal diseases could become one Alexander Gottschalk with the nema- nelrhodopsin-2 works like an “on” of the most promising clinical applica-

tode C. elegans, incidentally – that switch, halorhodopsin can be used tions of optogenetics. Photo:

24 MaxPlanckResearch 4 | 14 FOCUS_Optogenetics

2010

The colored light excites certain nerve cells in the brain of a rat. Researchers essentially activate or deactivate nerve cells with the touch of a button, allowing them to study for the first time what effects individual nerve cells have. This, in turn, makes it possible to study the network of billions of brain cells in intact organisms. Using gene technology methods, the opsins can be incorporated in very specific brain cells. It is thanks to this precision that optogenetics took hold so quickly; in 2010, the journal Nature Methods named it the »Method of the Year«.

» What appeared to be a caprice of nature is becoming a paradigm for the interplay between light and life.

TO THE POINT GLOSSARY

● In the early 1970s, Dieter Oesterhelt was the : This causes an electrical stimulus to be transmitted by first to discover, in Halobacterium salinarum, changing the membrane potential – that is, the electrical potential across the a retinal, the pigment that, at the time, was membrane of a nerve cell. known only from the retina of vertebrates. Archaebacteria, also known as : Single-celled organisms that emerged Retinal is a component in a membrane pro- very early on in evolution and that have usually adapted to extreme habitats. tein, bacteriorhodopsin. This is a light-driven In some properties, such as the lack of a nucleus, they are more like bacteria, proton pump that the bacterium uses to and in others, more like eukaryotes. convert sunlight to chemical energy – a new form of photosynthesis. : The lipid bilayer of biological membranes is impermeable to charged molecules, so also to ions. Ion channels are proteins that extend

● In 1980, the first indications were found that through the membrane and allow electrically charged particles to cross through. also the red eyespot apparatus of the green The transport takes place along an existing electrochemical gradient, the con- alga Chlamydomonas reinhardtii contains centration gradient. rhodopsin. In 2002, Peter Hegemann and Ion pump: Transmembrane proteins that regulate the transport of certain ions Georg Nagel were able to prove beyond any through a biological membrane are referred to as ion pumps. In contrast to doubt that this rhodopsin is the first example ion channels, they facilitate the of ions using energy. In this of a light-gated ion channel that is used to way, differences in the concentration of ions between the two sides of the control the flagellar beat. The researchers membrane can be maintained. called this novel protein channelrhodopsin. Optogenetics: A relatively new field that deals with controlling genetically mod- ified cells using light. It is based on light-activatable membrane proteins, such ● Various research groups succeeded in intro- as bacteriorhodopsin and channelrhodopsin, built into, for instance, nerve cells. ducing the gene sequences of these mem- brane proteins into nerve cells and express- Prokaryotes: Single-celled organisms whose DNA is not present in a cell nucle- ing it there. This made it possible for the us; these include bacteria and archaebacteria. Prokaryotes are distinguished first time to influence neural activity using from eukaryotes, organisms having a nucleus. light-switched channels or pumps in the Rhodopsin: A light-sensitive protein that contains retinal as a light-absorbing membrane – and so optogenetics was born. pigment. Photo: Karl Deisseroth, Viviana Gradinaru and John Carnett

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