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Light-activated enzymes for novel optogenetic approaches

In a project funded by the German Research Foundation (DFG), researchers from the Max Planck Institute for Medical Research in Heidelberg are studying algal and bacterial photoreceptors that can be used as optogenetic tools for studying regulatory and metabolic pathways. Through the insertion of a light-activated bacterial enzyme into zebrafish, the researchers have been able to visualise a hormonal reaction chain that is induced by stress factors.

Optogenetics uses techniques from genetics and optics for introducing photosensors into cells and controlling them with optical signals. Over the past ten years, optogenetics has virtually revolutionised the field of brain research; optogenetic techniques can be used to represent neurons and their numerous branches and connections in complex neural networks in a spatially and temporally precise manner. Moreover, neural activity can now be closely monitored, something that has previously only been possible in special cases in individual cells using the patch clamp technique.

Channelrhodopsins were discovered in 2003 and are the most common light receptors used in optogenetic investigations. Channelrhodopsins are light-gated ion pumps found in the cell membranes of the unicellular green algae, Chlamydomonas. They convert light energy into an electrochemical membrane potential, which the cells achieve by transporting ions across the membrane. The membrane potential delivers the power required by cells to operate other transport processes or to synthesise ATP. However, optogenetic methods are not only used in brain research and not limited to the use of light-gated ion channels.

A light-activated bacterial enzyme in zebrafish

Dr. Soojin Ryu, Max Planck Institute for Medical Research, Heidelberg. © MPImF

Dr. Soojin Ryu, head of the research group “Developmental Genetics of the Nervous System” at the Max Planck Institute for Medical Research in Heidelberg, has created transgenic zebrafish larvae in order to study stress-induced hormonal (endocrine) signalling chains. The transgenic zebrafish contain new channelrhodopsin variants, a light-activated adenylyl cyclase enzyme and light-gated G protein-coupled receptors (GPCR). Adenylyl cyclases are enzymes that catalyse the conversion of ATP to cyclic AMP (cAMP; adenosine monophosphate). They are activated by GPCRs once the latter have bound to their specific ligand (i.e. hormone).

Fish and humans react to stress in a similar way. Stress is a physical response that occurs as a direct effect of environmental impacts causing an upset in the body’s homeostasis. Stressors lead to the expression of two hormones – vasopressin and corticotropin-releasing hormone – in specific neurons in the hypothalamus, leading to the cAMP-mediated secretion of ACTH (adrenocorticotropic hormone) by the pituitary gland (hypophysis). ACTH in turn stimulates the secretion of corticosteroids such as cortisol. Corticosteroids are involved in a wide range of physiological processes in the central nervous system, cardiovascular system and metabolism. 

Dr. Ryu and her team inserted a light-activated Beggiatoa (ed. note: soil bacterium) adenylyl cyclase (bPAC) gene into the pituitary gland cell DNA of zebrafish larvae and succeeded in upregulating the corticosteroid level in the fish. Illuminating bPAC with blue light pulses, the researchers managed to turn enzyme activity on and off as well as modulate cAMP concentration and cortisol level; the animals too changed their response to stress in relation to cortisol concentration. The transgenic bPAC zebrafish larvae are excellent models for studying the individual steps of complex neuroendocrine processes and the behaviour of vertebrates in situations of stress. These investigations by the Max Planck scientists have expanded the use of optogenetic methods involving light-gated ion channels in nerve cell membranes to receptors such as GPCRs and intracellular enzymes such as bPAC. 

Protein-based light switches as optogenetic tools

Schematic showing the transfer of blue light signals between BLUF light receptors (grey) and effectors of the BLUF signalling cascade. © MPImF
Dr. Ryu’s research team is a member of the DFG-funded “Protein-based photoswitches as optogenetic tools” (FOR 1279) research group which involves scientists from seven German research institutions. The group’s objective is to advance the development of new optogenetic tools for applications in cell biology and the neurosciences. The “Photoreceptors” research group led by Professor Dr. Ilme Schlichting, head of the Department of Biomolecular Mechanisms and director at the Max Planck Institute for Medical Research in Heidelberg, is also part of FOR 1279. Schlichting, an internationally renowned biophysicist and protein structure expert, has for many years been focussing on light-activated enzymes and their photosensor domains, including rhodopsins and bPAC. Rhodopsins, which belong to a huge class of proteins in photoreceptor cells of the retina, contain retinal, a vitamin A-related carotinoid. The bPAC chromophore consists of flavin adenine dinucleotide (FAD).

Looking for new optogenetic tools

Prof. Dr. Ilme Schlichting © MPImF

Prof. Schlichting is particularly interested in blue light photoreceptors that use flavin-based photosensors. On the one hand, the researchers are studying proteins containing BLUF (“blue light sensor using FAD”) domains found in bacteria such as Beggiatoa as well as in unicellular organisms such as Euglenozoa, and fungi. On the other hand, Schlichting’s team is also studying light-activated kinases (phototropins) of Mougeotia green algae and light-regulated transcription factors (aureochromes) of gold algae such as Vaucheria. These proteins contain so-called LOV (“light-oxygen-voltage-sensing”) domains, whose structure is different from that of the BLUF domains, but also contain flavin chromophores.

Under the FOR 1279 project, Schlichting and her team are determining the crystallographic structures of the aforementioned light-activated proteins and subsequently carrying out detailed enzymatic, spectroscopic and functional studies both in vitro and in vivo. The scientists are also undertaking comparative studies with synthetic systems such as genetically fused rhodopsin-cyclase chimeras, thereby providing science with a complete set of light-controlled tools with different, well characterised properties for optogenetic examinations. In addition to being used for elucidating neuroscientific issues, these tools are also excellently suited to investigating regulatory processes (cell and developmental biology) and metabolic pathways in healthy and diseased organisms. 

References:
DeMarco RJ, Groneberg AH, Yeh C-M, Castillo Ramirez LA, Ryu S (2013): Optogenetic elevation of endogenous glucocorticoid level in larval zebrafish. Frontiers in Neural Circuits 7, 1-11.
Jung A, Domratcheva T, Schlichting I: Detaillierte Einblicke in die Umwnadlung von Blaulichtsignalen. Research Report from Webservice 2007. Max Planck Institute for Medical Research.

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