Curated Optogenetic Publication Database

Abstract: Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Abstract: The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Abstract: Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Abstract: In neural mechanisms of animal learning, intracellular cAMP has been known to play an important role. In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into Aplysia neurons, and explored whether PAC can produce cAMP in the neurons by light stimulation. Serotonergic modulation of mechanoafferent sensory neurons in Aplysia pleural ganglia has been reported to increase intracellular cAMP level and promotes synaptic transmission to motor neurons by increasing spike width of sensory neurons. When cAMP was directly injected into the sensory neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was not substituted by injection of 5'AMP, and maintained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of sensory neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.

Abstract: Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.