Functional transplant of photoactivated adenylyl cyclase (PAC) into Aplysia sensory neurons

Nagahama, Tatsumi; Suzuki, Takeshi; Yoshikawa, Shinya; Iseki, Mineo

doi:10.1016/j.neures.2007.05.015

Cited by 52 publications

(31 citation statements)

References 28 publications

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“…Such a sharp switching property of PAC is suitable to be used as a tool to control various cellular processes by light, i.e., optogenetics. In fact, attempts to optogeneticically control cAMP levels in Aplysia neurons (Nagahama et al 2007), Xenopus oocytes, cultured mammalian cells, Drosophila brains (Schröder-Lang et al 2007), and Caenorhabditis neurons (Stierl et al 2011;Weissenberger et al 2011) have been reported.…”

Section: −2mentioning

confidence: 99%

Photomovement in Euglena

Häder

Iseki

2017

Advances in Experimental Medicine and Biology

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Motile microorganisms such as the green Euglena gracilis use a number of external stimuli to orient in their environment. They respond to light with photophobic responses, photokinesis and phototaxis, all of which can result in accumulations of the organisms in suitable habitats. The light responses operate synergistically with gravitaxis, aerotaxis and other responses. Originally the microscopically obvious stigma was thought to be the photoreceptor, but later the paraxonemal body (PAB, paraflagellar body) has been identified as the light responsive organelle, located in the trailing flagellum inside the reservoir. The stigma can aid in light direction perception by shading the PAB periodically when the cell rotates helically in lateral light, but stigmaless mutants can also orient with respect to the light direction, and negative phototaxis does not need the presence of the stigma. The PAB is composed of dichroically oriented chromoproteins which is reflected in a pronounced polarotaxis in polarized light. There was a long debate about the potential photoreceptor molecule in Euglena, including carotenoids, flavins and rhodopsins. This discussion was terminated by the unambiguous proof that the photoreceptor is a 400 kDa photoactivated adenylyl cyclase (PAC) which consists of two α- and two β-subunits each. Each subunit possesses two BLUF (Blue Light receptor Using FAD) domains binding FAD, which harvest the light energy, and two adenylyl cyclases, which produce cAMP from ATP. The cAMP has been found to activate one of the five protein kinase s found in Euglena (PK.4). This enzyme in turn is thought to phosphorylate proteins inside the flagellum which result in a change in the flagellar beating pattern and thus a course correction of the cell. The involvements of PAC and protein kinase have been confirmed by RNA interference (RNAi). PAC is responsible for step-up photophobic responses as well as positive and negative phototaxis, but not for the step-down photophobic response, even though the action spectrum of this resembles those for the other two responses. Analysis of several colorless Euglena mutants and the closely related Euglena longa (formerly Astasia longa) confirms the results. Photokinesis shows a completely different action spectrum. Some other Euglena species, such as E. sanguinea and the gliding E. mutabilis, have been investigated, again showing totally different action spectra for phototaxis and photokinesis as well as step-up and step-down photophobic responses.

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Section: −2mentioning

confidence: 99%

Photomovement in Euglena

Häder

Iseki

2017

Advances in Experimental Medicine and Biology

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“…The protein size, 350 amino acids, is much smaller than the sizes of ␣ or ␤ subunits of PAC from E. gracilis, which are approximately 1000 amino acids. This makes BlaC an attractive alternative to PAC, which is currently used in optogenetic applications involving manipulations of the cAMP levels (22,23).…”

Section: The Blac Protein From Beggiatoa Sp Ps Is a Blue Light-activmentioning

confidence: 99%

“…The BLUF-domain containing photoactivated adenylyl cyclase from Euglena gracilis, PAC (21), proved to be a powerful analytical tool to control cAMP levels in Xenopus oocytes and neurons of Drosophila and the mollusk Aplysia (22,23). PAC proved useful despite the large size of its subunits (Ͼ1000 amino acids), difficulties in heterologous expression (24), and high background activity in the dark when expressed in vivo (22).…”

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confidence: 99%

Natural and Engineered Photoactivated Nucleotidyl Cyclases for Optogenetic Applications

Ryu

Moskvin

Siltberg-Liberles

et al. 2010

Journal of Biological Chemistry

205

239

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Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and upregulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.

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“…Hence, the light-induced amount of cAMP produced by euPACα in cholinergic neurons directly affected the intracellular signaling and caused changes in the swimming frequency and the speed of locomotion (Weissenberger et al 2011). Furthermore, blue light stimulation of euPACα has been shown to increase the spike width in Aplysia sensory neurons (Nagahama et al 2007). In Drosophila larvae that expressed euPACα in motor neurons, miniature excitatory junction potential (mEJP) frequency was highly increased after blue light irradiation (Bucher and Buchner 2009).…”

Section: Application Of Light-sensitive Modules In Synthetic Biology mentioning

confidence: 99%