A PRIMER ON OPTOGENETICS
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Optogenetics: Teaching dark cells to see the light
Which processes can be controlled by light? What can I do with optogenetics? Are there applications in industry research and in therapy?
Optogenetics: Teaching dark cells to see the lightLight signals regulate essential physiological and behavioural processes in most organisms (for instance, simple forms of movement in microbes, photosynthesis in plants or vision in animals). Since the early 2000s, research methods have been developed that repurpose these natural light sensing capabilities to control living cells that are normally not responsive to light (e.g., specific nerve cells buried deep in the brain of a laboratory animal). The motivation for these developments was that manipulation by light offers unique advantages over existing strategies to manipulate biological systems (such as pharmacology, i.e. drugs, or genetics, e.g., genome editing using CRISPR). Light acts in real-time and permits not only unparalleled temporal precision (e.g., to target processes during a specific behaviour or during a selected stage in development) but also spatial precision (e.g., to target selected compartments in a cell or selected cells in a tissue). Also, light can be readily applied to and withdrawn from a broad variety of biological samples, and, finally, the application of light can be inexpensive. As these methods are achieved by genetic targeting of light-sensing proteins into specific cells, they have been collectively termed 'optogenetics'. The emerging field of optogenetics is highly interdisciplinary as it builds on fundamental photobiology, applies protein engineering and utilizes advanced instrumentation and gene delivery techniques to ultimately look at the behavior of cells and animals in health and disease. This diversity is also reflected in the Australian research community.
The past decade have seen a spectacular increase in the molecular and cellular processes that can be controlled by light. Prominent examples include gene transcription, subcellular protein localization and a variety of cell signaling pathways. Light control in optogenetics is generally achieved using microbial and plant photoreceptor domains that are in this context referred to as 'optogenetic tools'. Photon absorption coverts these proteins from the so called 'dark state' or resting state to the 'lit state' or 'light state' that in many cases is functional or catalytically-active. Most optogenetic tools operate in one of two modes. In the first mode, a natural photoreceptor function (e.g., transmembrane ion flow in case of microbial opsins such as channelrhodopsin or enzyme activity in case of the photoactivated cyclases) is harnessed to control cell behavior. In the second mode, light-regulated inter- or intramolecular (un)binding reactions, which occur in the specific regulatory domains of photoreceptors are engineered into modular multi-domain proteins (e.g., in the case of the crytochrome clustering or heterodimerization). These are further optimized, e.g., with respect to achieving high expression or diverse steady-state and dynamic properties. Light-activated ion channels (e.g., channelrhodopsins or Cl-/H+ pumps) are now a part of the toolbox of nearly every neurobiologist, whereas light-activated enzymes and gene manipulation techniques are actively developed in cell biology.
The impact and potential of optogenetics is widely recognized:
Method of the Year
of Nature Methods in 2010
Top 10 Emerging Technology
at the 2016 World Economic Forum
What can I do with optogenetics?
Optogenetics is uniquely suited to answer fundamental questions in virtually all fields of biology. Indeed the technique has been applied in vitro and in vivo to cells of the central and peripheral nervous systems, muscle cells or stem cells in rodents, fish, flies and worms as models of health and disease.
The strengths of optogenetics in controlling biological processes are:
1. High spatial precision (down to micrometers, e.g., in two-photon activation)
2. High temporal precision (down to milliseconds) and reversibility
3. Tunable activation strength (signal magnitude depends on light intensity)
4. Selectivity for a target protein pathway through genetic modification by photoreceptors
5. Non-invasive 'remote control'
6. Light can be an inexpensive stimulus
Based on these strengths, questions such as these can be addressed:
What is the function of individual cells or a specific cell type in a complex tissue (e.g., in a brain circuit)?
At what point in time during development is a certain signaling pathway required (e.g., for morphogenesis)?
Does the 'front' of a cell respond differently to the 'back' of a cell (e.g., during directed cell migration)?
Which signaling pathway is sufficient to trigger a behavior in situ (e.g., during tissue regeneration)?
How does a certain activity pattern during a 'playback' affect cellular reponses (e.g., to confirm a model or simulation)?
Further reading on the application of optogenetics in cell signaling:
Are there applications in industry research and in therapy?
Optical control and optogenetic technologies have matured to the point where they are applied in commerical research and therapy. One prominent example for commerical research is the use of light-activated ion channels in high-throughput drug discovery. In these experiments pioneered by Axxam SpA (Milan, Italy) and Q-State Bioscience (Cambridge, MA, USA), light depolarizes cells that express voltage-gated ion channel drug targets. Notably, depolarization and simultanenous detection of membrane voltage is performed in the 384-well plate format and without the need of electrophysiology or solution exchange. This 'all-optical' experimental paradigm enables high-content screening on new scales and has high general potential in the study of cell signaling and cell behavior. The pioneering application of optogenetics as a therapy tackles the restoration of vision in patients with hereditary retinal degeneration. GenSight Biologics (Paris, France) and Allergan plc (Dublin, Ireland) are currently conducting clinical trials in which channelrhodopsin variants are virally delivered into the retinas of patients suffering from retinitis pigmentosa.