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    Department of Botany I - Plant-Physiology and Biophysics

    Light-activated Cyclases: PAC & Cyclop

    Already in 2002, a Japanese working group discovered light-activated adenylyl cyclases in the monocellular flagellate Euglena gracilis (Iseki et al., 2007, Nature) which they named photoactivated adenylyl cyclase (PAC) a and PACb. The use of PAC from Euglena (EuPAC) as an optogenetic tool in animal cells and the fruit fly Drosophila melanogaster (by Martin Schwärzel, now FU Berlin) was shown by us in collaboration with other groups in 2007 (Schröder-Lang et al., Nature Methods) , See fig. 1.

    Other PACs were found in genome data from microbes and were characterized by us and others. Particularly attractive is PAC from the bacterium Beggiatoa spec. (bPAC), since it is only 350 amino acids in size and has a high ratio of activity in light to activity in the dark (L / D). By mutagenesis and gene fusion we are currently changing and improving the properties of bPAC.

    In 2014, a Brazilian group showed that a rhodopsin fusion with a guanylyl cyclase is expressed in the aquatic fungus Blastocladiella emersonii and is activated by green light (Avelar et al., 2014, Curr. Biol.). We synthesized the DNA and characterized the protein after heterologous expression in oocytes or HEK293 cells. After extensive characterization (Gao et al., 2015, Nat. Commun.), We named the protein Cyclase Opsin or Cyclop, and also showed light-activated inward currents after coexpression of a cGMP-sensitive cation channel, see Fig. 2.

    We are currently working on gene fusions of Cyclop and mutations to produce an adenylyl cyclase from the guanylyl cyclase: Cyclop-PAC.

    The figure shows the schematic of an oocyte with measuring electrodes and heterologously expressed proteins on the left (A),
    Heterologous expression of PAC allows rapid concentration increase of cAMP in cells on exposure to light, as measured by cAMP-sensitive ion channels on-line, see scheme in A.
    (Graphic: G. Nagel, Universität Würzburg 2017)
    and 2 conductivity traces of an oocyte in which a cAMP-sensitive anion channel (CFTR)
    The figure shows that the conductivity oft he same cell can be achieved either by administering IBMX and forskolin during time indicated by bar (increase of cAMP via endogenous enzymes, see B) or by exposure to light during time indicated by bar (activation of PAC, see C).
    (Graphic: G. Nagel, Universität Würzburg 2017)
    The figure shows left (A) the scheme of an oocyte with heterologously expressed Cyclop and a CNG cation channel. To the right is a current trace in B,
    Heterologous expression of Cyclop allows rapid concentration increase of cGMP. A: Scheme of an oocyte with heterologously expressed light-sensitive Cyclop and CNG cation channel. B: Fast activation of a CNG-mediated inward current after a light flash of 100 ms duration.
    (Graphic: G. Nagel, Universität Würzburg 2017)
    as well as the action spectrum of Cyclop with maximum at approx. 530 nm in C.
    C: Action spectrum of light-stimulated cGMP generation by Cyclop.
    (Graphic: G. Nagel, Universität Würzburg 2017)
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