Peter Hegemann - Academia.edu (original) (raw)

Papers by Peter Hegemann

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alg... more The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin- deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green

Proceedings of the National Academy of Sciences, 2014

Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As l... more Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

Biophysical Journal, 2001

Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via... more Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via generation of photocurrents across the plasma membrane. Chlamydomonas mutants blocked in retinal biosynthesis are “blind,” but they can be rescued by the addition of exogenous retinoids. Photosignaling by chlamyrhodopsin regenerated with 9-demethylretinal was investigated by recording photocurrents from single cells and cell suspensions, and by measuring phototactic orientation.

Nature Neuroscience, 2008

would maintain the important property of chemical cofactor independence in mammalian brains. Howe... more would maintain the important property of chemical cofactor independence in mammalian brains. However, it has been unclear how such a fundamental new property could be achieved with channelrhodopsins and how the resulting stable depolarization could be precisely terminated as needed at a specified time, particularly if the offset of depolarization is no longer coupled to the offset of the triggering light pulse itself.

Photochemistry and Photobiology, 1988

... and the sensory rhodopsin from Halobacterium halobium (Lanyi and Schobert, 1983) can be bleac... more ... and the sensory rhodopsin from Halobacterium halobium (Lanyi and Schobert, 1983) can be bleached with hydroxylamine at concentrations in the same range as necessary for Chlamydomonus rhodopsin. Unfortunately, data for bleaching these rhodopsins in whoie ceits ...

Proceedings of the National Academy of Sciences of the United States of America, 2011

Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise indu... more Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-tocysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for lightcontrolled activation of neurons. hippocampus | optogenetics | photocycle | pyramidal cell | spike frequency

Frontiers in Molecular Neuroscience , 2013

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here ... more Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca 2+ -bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca 2+ ]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca 2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Genome Biology, 2013

The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both bi... more The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Proceedings of The National Academy of Sciences, 2006

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photorecept... more BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather

The Plant Journal, 1999

The use of Chlamydomonas reinhardtii as a model system has been hindered by dif®culties encounter... more The use of Chlamydomonas reinhardtii as a model system has been hindered by dif®culties encountered in expressing foreign genes. We have synthesised a gene encoding green¯uorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-¯uorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became¯uorescent indicating that the opsin± GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodi®ed opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

THE PLANT CELL ONLINE, 2008

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors i... more Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H þ -channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H þ , CHR1 also conducts Na þ , K þ , and Ca 2þ . The kinetic selectivity analysis demonstrates that H þ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1 505 and CHR1 463 , that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

THE PLANT CELL ONLINE, 1999

Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls t... more Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls the organism's phototactic behavior via two independent photoreceptor currents. Here, we report the identification of an opsinlike gene, designated as volvoxopsin ( vop ). The encoded protein exhibits homologies to the opsin of the unicellular alga Chlamydomonas reinhardtii (chlamyopsin) and to the entire animal opsin family, thus providing new perspectives on opsin evolution. Volvoxopsin accumulates within the eyes of somatic cells. However, the vop transcript is detectable only in the reproductive eyeless gonidia and embryos. vop mRNA levels increase 400-fold during embryogenesis, when embryos develop in darkness, whereas the vop transcript does not accumulate when embryos develop in the light. An antisense transformant, T3, was generated. This transformant produces 10 times less volvoxopsin than does the wild type. In T3, the vop transcript is virtually absent, whereas the antisense transcript is predominant and light regulated. It follows that vop expression is under light-dependent transcriptional control but that volvoxopsin itself is not the regulatory photoreceptor. Transformant T3 is phototactic, but its phototactic sensitivity is reduced 10-fold relative to the parental wild-type strain HK10. Thus, we offer definitive genetic evidence that a rhodopsin serves as the photoreceptor for phototaxis in a green alga. Holland, E.-M., Braun, F.-J., Nonnengässer, C., Harz, H., and Hegemann, P. (1996). The nature of rhodopsin triggered photocurrents in Chlamydomonas. I. Kinetics and influence of divalent ions. Biophys. J. 70, 924-931. and molecular approaches to Volvox development and evolution. Int. Rev. Cytol. 99, 217-293. Kirk, D.L., Kaufman, M.R., Keeling, R.M., and Stamer, K.A. (1991). Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo. Development 1 (suppl.), 67-82. Kirk, M.M., and Kirk, D.L. (1985). Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development. Cell 41, 419-428.

Science, 2002

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-typ... more Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

Plant Molecular Biology, 2004

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in r... more For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .

Photochemistry and Photobiology, 2009

Channelrhodopsins (ChR1 and ChR2) are directly light-gated ion channels acting as sensory photore... more Channelrhodopsins (ChR1 and ChR2) are directly light-gated ion channels acting as sensory photoreceptors in the green alga Chlamydomonas reinhardtii. These channels open rapidly after light absorption and both become permeable for cations such as H(+), Li(+), Na(+), K(+) and Ca(2+). K(m) for Ca(2+) is 16.6 mm in ChR1 and 18.3 mm in ChR2 whereas the K(m) values for Na(+) are higher than 100 mm for both ChRs. Action spectra of ChR1 peak between 470 and 500 nm depending on the pH conditions, whereas ChR2 peaks at 470 nm regardless of the pH value. Now we created two chimeric ChRs possessing helix 1-5 of ChR1 and 6, 7 of ChR2 (ChR1/2(5/2)), or 1, 2 from ChR1 and 3-7 from ChR2 (ChR1/2(2/5)). Both ChR-chimera still showed pH-dependent action spectra shifts. Finally, a mutant ChR1E87Q was generated that inactivated only slowly in the light and showed no spectral shift upon pH change. The results indicate that protonation/deprotonation of E87 in helix 1 alters the chromophore polarity, which shifts the absorption and modifies channel inactivation accordingly. We propose a trimodal counter ion complex for ChR1 but only a bimodal complex for ChR2.

Photochemistry and Photobiology, 2014

The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 fr... more The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii was studied by absorption and fluorescence spectroscopy. The retinal cofactor of HKR1 exists in two Schiff base forms RetA and RetB. RetA is the deprotonated 13-cis-retinal Schiff base (RSB) absorbing in the UVA spectral region. RetB is the protonated all-trans RSB absorbing in the blue spectral region. Blue light exposure converts RetB fully to RetA. UVA light exposure converts RetA to RetB and RetB to RetA giving a mixture determined by their absorption cross sections and their conversion efficiencies. The quantum efficiencies of conversion of RetA to RetB and RetB to RetA were determined to be 0.096 ± 0.005 and 0.405 ± 0.01 respectively. In the dark thermal equilibration between RetA and RetB with dominant RetA content occurred with a time constant of about 3 days at room temperature. The fluorescence emission behavior of RetA and RetB was studied, and fluorescence quantum yields of ϕ(F) (RetA) = 0.00117 and ϕ(F) (RetB) = 9.4 × 10(-5) were determined. Reaction coordinate schemes of the photodynamics are developed.

Photochemistry and Photobiology, 2002

The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen-and vol... more The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen-and voltage-sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild-type (LOV1-WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1-C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of F ‫؍‬ 0.30 and a fluorescence lifetime of F ‫؍‬ 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about F ‫؍‬ 0.17 and F ‫؍‬ 2.9 ns for LOV1-WT, indicating an enhanced intersystem crossing in LOV1-WT because of the adjacent sulfur of C57. A single-exponential fluorescence decay was observed in picosecond laser time-resolved fluorescence measurements for both LOV1-WT and LOV1-C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady-state fluorescence measurements for LOV1-WT, which is thought to be because of the formation of a long-lived intermediate flavin-C(4a)-cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1-C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.

Photochemical & Photobiological Sciences, 2004

ABSTRACT

Nucleic Acids Research, 2005

The success of long polynucleotide de novo synthesis is largely dependent on the quality and puri... more The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes--T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V--have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.

Nature Neuroscience, 2008

The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin ... more The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type-specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted ~70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alg... more The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin- deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green

Proceedings of the National Academy of Sciences, 2014

Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As l... more Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

Biophysical Journal, 2001

Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via... more Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via generation of photocurrents across the plasma membrane. Chlamydomonas mutants blocked in retinal biosynthesis are “blind,” but they can be rescued by the addition of exogenous retinoids. Photosignaling by chlamyrhodopsin regenerated with 9-demethylretinal was investigated by recording photocurrents from single cells and cell suspensions, and by measuring phototactic orientation.

Nature Neuroscience, 2008

would maintain the important property of chemical cofactor independence in mammalian brains. Howe... more would maintain the important property of chemical cofactor independence in mammalian brains. However, it has been unclear how such a fundamental new property could be achieved with channelrhodopsins and how the resulting stable depolarization could be precisely terminated as needed at a specified time, particularly if the offset of depolarization is no longer coupled to the offset of the triggering light pulse itself.

Photochemistry and Photobiology, 1988

... and the sensory rhodopsin from Halobacterium halobium (Lanyi and Schobert, 1983) can be bleac... more ... and the sensory rhodopsin from Halobacterium halobium (Lanyi and Schobert, 1983) can be bleached with hydroxylamine at concentrations in the same range as necessary for Chlamydomonus rhodopsin. Unfortunately, data for bleaching these rhodopsins in whoie ceits ...

Proceedings of the National Academy of Sciences of the United States of America, 2011

Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise indu... more Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-tocysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for lightcontrolled activation of neurons. hippocampus | optogenetics | photocycle | pyramidal cell | spike frequency

Frontiers in Molecular Neuroscience , 2013

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here ... more Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca 2+ -bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca 2+ ]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca 2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Genome Biology, 2013

The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both bi... more The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Proceedings of The National Academy of Sciences, 2006

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photorecept... more BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather

The Plant Journal, 1999

The use of Chlamydomonas reinhardtii as a model system has been hindered by dif®culties encounter... more The use of Chlamydomonas reinhardtii as a model system has been hindered by dif®culties encountered in expressing foreign genes. We have synthesised a gene encoding green¯uorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-¯uorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became¯uorescent indicating that the opsin± GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodi®ed opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

THE PLANT CELL ONLINE, 2008

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors i... more Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H þ -channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H þ , CHR1 also conducts Na þ , K þ , and Ca 2þ . The kinetic selectivity analysis demonstrates that H þ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1 505 and CHR1 463 , that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

THE PLANT CELL ONLINE, 1999

Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls t... more Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls the organism's phototactic behavior via two independent photoreceptor currents. Here, we report the identification of an opsinlike gene, designated as volvoxopsin ( vop ). The encoded protein exhibits homologies to the opsin of the unicellular alga Chlamydomonas reinhardtii (chlamyopsin) and to the entire animal opsin family, thus providing new perspectives on opsin evolution. Volvoxopsin accumulates within the eyes of somatic cells. However, the vop transcript is detectable only in the reproductive eyeless gonidia and embryos. vop mRNA levels increase 400-fold during embryogenesis, when embryos develop in darkness, whereas the vop transcript does not accumulate when embryos develop in the light. An antisense transformant, T3, was generated. This transformant produces 10 times less volvoxopsin than does the wild type. In T3, the vop transcript is virtually absent, whereas the antisense transcript is predominant and light regulated. It follows that vop expression is under light-dependent transcriptional control but that volvoxopsin itself is not the regulatory photoreceptor. Transformant T3 is phototactic, but its phototactic sensitivity is reduced 10-fold relative to the parental wild-type strain HK10. Thus, we offer definitive genetic evidence that a rhodopsin serves as the photoreceptor for phototaxis in a green alga. Holland, E.-M., Braun, F.-J., Nonnengässer, C., Harz, H., and Hegemann, P. (1996). The nature of rhodopsin triggered photocurrents in Chlamydomonas. I. Kinetics and influence of divalent ions. Biophys. J. 70, 924-931. and molecular approaches to Volvox development and evolution. Int. Rev. Cytol. 99, 217-293. Kirk, D.L., Kaufman, M.R., Keeling, R.M., and Stamer, K.A. (1991). Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo. Development 1 (suppl.), 67-82. Kirk, M.M., and Kirk, D.L. (1985). Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development. Cell 41, 419-428.

Science, 2002

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-typ... more Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

Plant Molecular Biology, 2004

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in r... more For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .

Photochemistry and Photobiology, 2009

Channelrhodopsins (ChR1 and ChR2) are directly light-gated ion channels acting as sensory photore... more Channelrhodopsins (ChR1 and ChR2) are directly light-gated ion channels acting as sensory photoreceptors in the green alga Chlamydomonas reinhardtii. These channels open rapidly after light absorption and both become permeable for cations such as H(+), Li(+), Na(+), K(+) and Ca(2+). K(m) for Ca(2+) is 16.6 mm in ChR1 and 18.3 mm in ChR2 whereas the K(m) values for Na(+) are higher than 100 mm for both ChRs. Action spectra of ChR1 peak between 470 and 500 nm depending on the pH conditions, whereas ChR2 peaks at 470 nm regardless of the pH value. Now we created two chimeric ChRs possessing helix 1-5 of ChR1 and 6, 7 of ChR2 (ChR1/2(5/2)), or 1, 2 from ChR1 and 3-7 from ChR2 (ChR1/2(2/5)). Both ChR-chimera still showed pH-dependent action spectra shifts. Finally, a mutant ChR1E87Q was generated that inactivated only slowly in the light and showed no spectral shift upon pH change. The results indicate that protonation/deprotonation of E87 in helix 1 alters the chromophore polarity, which shifts the absorption and modifies channel inactivation accordingly. We propose a trimodal counter ion complex for ChR1 but only a bimodal complex for ChR2.

Photochemistry and Photobiology, 2014

The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 fr... more The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii was studied by absorption and fluorescence spectroscopy. The retinal cofactor of HKR1 exists in two Schiff base forms RetA and RetB. RetA is the deprotonated 13-cis-retinal Schiff base (RSB) absorbing in the UVA spectral region. RetB is the protonated all-trans RSB absorbing in the blue spectral region. Blue light exposure converts RetB fully to RetA. UVA light exposure converts RetA to RetB and RetB to RetA giving a mixture determined by their absorption cross sections and their conversion efficiencies. The quantum efficiencies of conversion of RetA to RetB and RetB to RetA were determined to be 0.096 ± 0.005 and 0.405 ± 0.01 respectively. In the dark thermal equilibration between RetA and RetB with dominant RetA content occurred with a time constant of about 3 days at room temperature. The fluorescence emission behavior of RetA and RetB was studied, and fluorescence quantum yields of ϕ(F) (RetA) = 0.00117 and ϕ(F) (RetB) = 9.4 × 10(-5) were determined. Reaction coordinate schemes of the photodynamics are developed.

Photochemistry and Photobiology, 2002

The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen-and vol... more The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen-and voltage-sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild-type (LOV1-WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1-C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of F ‫؍‬ 0.30 and a fluorescence lifetime of F ‫؍‬ 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about F ‫؍‬ 0.17 and F ‫؍‬ 2.9 ns for LOV1-WT, indicating an enhanced intersystem crossing in LOV1-WT because of the adjacent sulfur of C57. A single-exponential fluorescence decay was observed in picosecond laser time-resolved fluorescence measurements for both LOV1-WT and LOV1-C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady-state fluorescence measurements for LOV1-WT, which is thought to be because of the formation of a long-lived intermediate flavin-C(4a)-cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1-C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.

Photochemical & Photobiological Sciences, 2004

ABSTRACT

Nucleic Acids Research, 2005

The success of long polynucleotide de novo synthesis is largely dependent on the quality and puri... more The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes--T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V--have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.

Nature Neuroscience, 2008

The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin ... more The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type-specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted ~70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.