Still using flashlights for optogenetic research?

What the heck is optogenetics?

What is Optogenetics?


Image

Optogenetics is a biological technology for controlling cellular activity with light. More specifically, the technology is a combination of methods from optics and genetics, with the aim of switching on (gain-of-function) or off (loss-of-function) certain functional events in specific cells or living tissues. For this purpose, foreign genes are introduced via viral vectors into the target cells that lead to the expression of light-sensitive proteins. Upon illumination, these optogenetic proteins undergo a conformational change that subsequently triggers a cellular response.

Some photoactivatable proteins contain an intrinsic signaling component that can be triggered by light. These 'first generation' optogenetic proteins, such as channelrhodopsin, are hardwired to a certain signal transduction pathway. More recently developed optogenetic tools overcome this limitation by artificially fusing light-responsive proteins or their interaction partners to different target signaling output domains. This method has huge advantages and gives rise to the precise temporal and spatial control of virtually any cellular signaling pathway by a simple flash of light.

Neuronal Optogenetics - Controlling the Brain

Optogenetics is most commonly perceived as a neuromodulation technique targeting the brain. Indeed, one of the first applications of an optogenetic tool, the expression of channelrhodopsin in neurons, allowed for the first time the precise control of neuronal activity solely with light. Channelrhodopsin is a light-gated ion channel that conducts cation influx upon illumination with blue light. Precisely, the introduction of this single-component optogenetic system into neuronal cells leads to light-induced depolarization of the cell membrane and hence the induction of action potentials.

Shortly after this discovery, a light-gated ion pump was added to the optogenetic toolbox. Upon green/yellow illumination, halorhodopsin pumps chloride ions into the cell, leading to hyperpolarization of the cell membrane. Artificial introduction of halorhodopsin into neurons can thus be used to optogenetically inhibit action potentials and thus silence neuronal activity of the brain.

Another more complex tool to inhibit neuronal activity is based on the light-sensitive receptor protein rhodopsin. This G protein coupled receptor (GPCR) acts through the recruitment of intracellular signaling components, ultimately silencing neuronal action potentials.

Optogenetic control of neuronal activity has thus far led to numerous new findings in the fields of neuroscience and neuropsychiatric disease. Among those neural circuits investigated so far are brain disorders such as Parkinson's disease, schizophrenia, autism, drug abuse, depression and anxiety.

Non-Neuronal Optogenetics - Unraveling Cell Signaling

After the successful application in neurosciences, it did not take long to discover that the characteristics of optogenetics are also advantageous in other areas of biology. The field of non-neuronal optogenetics relies on 'second generation' light-responsive fusion proteins that are assembled in a modular fashion. These genetically-encoded photoactivatable proteins are not hardwired to a specific cellular response but can be fused to virtually any output or signaling domain. Non-neuronal optogenetics allows for the precise temporal and spatial control of a chosen signaling pathway of interest by irradiation of the cells. In addition, non-neuronal optogenetics has been successfully applied for the control of cellular motility, cell contraction, apoptosis and cell differentiation.

Optogenetic Approaches: How does Optogenetics work?


Image

Optogenetics makes use of a defined set of molecular approaches. In general, light stimulation leads to a conformational change within a photosensitive domain fused to an output domain. This leads either to the activation (gain-of-function) or deactivation (loss-of-function) of downstream signaling networks.

The most commonly used approach is the light-dependent recruitment of a protein of interest to the cell membrane. In this case, the protein of interest is tagged with a photosensitive domain that can bind to a specific anchor protein upon light activation. Membrane recruitment of for example SOS has been used to optogenetically activate the Raf/MEK/ERK signaling pathway in mammalian cells.

Using the same principal, it is also possible to sequester away a protein of interest from its designated site of action. A clustered bait protein removes the protein of interest from its natural target by interacting, in a light-dependent manner, with the fused photosensitive prey protein. Essentially, sequestering away the protein of interest from its natural localization for example into a different cellular compartment inactivates the initial function of the protein. This has previously been used to inactivate target proteins such as Guanine nucleotide exchange factors (GEFs) and GTPases like RhoG.

Another widely utilized optogenetic approach exploits the tendency of some photosensitive proteins to form large homomeric clusters when activated by light. If the target protein is catalytically active when present in high density, then clustering results in the formation of active signaling hubs inside the cell. This cluster-dependent activation of signaling was demonstrated to be useful in inducing localized actin polymerization.

Optogenetics can also be used to control gene expression. This approach usually makes use of light-driven dimerization or monomerization of photoactivatable proteins. When fused to either a split transcription factor or a transcriptional activator or repressor, these constructs allow the precise spatiotemporal activation or inhibition of transcription of a gene of interest.

Using a slightly different approach, it is also possible to directly control the enzymatic activity or binding affinity of a target protein. The strategy here is to bioengineer a light-activatable scaffold around the signaling motif of the protein of interest. Light irradiation leads to a conformational change within the photosensitive fusion domain and the subsequent exposure of the hidden signaling motif. Such photo-uncaging strategies have been used to trigger the exposure of degrons to cause protein degradation.

Common Optogenetic Proteins


Image

The optogenetic toolbox used in non-neuronal optogenetics consists of a variety of photoactivatable proteins and their interaction partners. Generally speaking, the underlying mechanism of most of these proteins is light-dependent monomerization or oligomerization. Until today, the range of optogenetic tools is constantly being expanded, either by genetically modifying and optimizing existing optogenetic proteins or by discovering entirely novel proteins or protein domains. They originate from all three domains of life and can thus be found in Archaea (such as halorhodopsin), Bacteria (such as bacteriophytochrome), and Eukaryota (such as channelrhodopsin).

The commonly used plant UV photoreceptor UVR8 forms a stable homodimer in the dark. Ultraviolet irradiation induces rapid disruption and dissociation of the dimer. Monomeric UVR8 is then able to bind to its interaction partner COP1, which activates the function of the fused protein of interest and its downstream signaling cascade. Relaxation of the system is achieved in the dark and can take several hours.

Another optogenetic tool found in plants is the flavin mononucleotide binding protein AsLOV2 with its C-terminal Jα alpha helix. Blue light irradiation causes the Jα alpha helix to swing out from the LOV core domain, revealing a previously hidden binding site for a downstream signaling protein. Photoreversion in the dark occurs on a timescale of minutes.

Blue light illumination of the plant cryptochrome CRY2 leads to photoactivation and subsequent binding to its interaction partner CIB1. Similar to the UVR8-COP1 system, this triggers a downstream signaling cascade from the fused protein of interest. Within minutes, the CRY2-CIB1 heterodimers dissociate in the dark, terminating the signal.

Among the optogenetic proteins, Dronpa occupies a special role. Not only can it reversibly switch between a tetrameric and a monomeric state by blue/green and violet light, respectively. Beyond that, it is also a switchable fluorescent protein, exhibiting strong fluorescence only in its oligomeric state. Dronpa has been used to cage fusion protein function in its oligomeric state. Monomerization after illumination exposes the active sites of these proteins of interest, which activates their initial cellular function.

Lastly, phytochromes are red/far-red light-responsive photoreceptors found in microbes and plants. The widely used PhyB/PIF optogenetic system from the model plant Arabidopsis thaliana makes use of the light-dependent interaction of the phytochrome PhyB with the phytochrome interacting factor PIF. Thus, fusion to output domains is used to reliably switch between active and inactive signaling states in the range of milliseconds to seconds.

Optogenetics TED Talk in Heidelberg


Optogenetic LED illumination

To regulate optogenetic tools (and all other optically controlled substances), they need to be illuminated with a specific wavelength. Although wavelength specific illumination devices are available (LED-flashlights etc.) they are often not suited for a particular experimental approach. We at opto biolabs develop illumination devices suited to your experimental approach. Have a look in our product section and find the right illumination device for your optogenetic cell system. Do not hesitate to contact us if you cannot find a suitable solution. We build customized illumination devices tailored to your problem.

Check out our pxONE here and get ready for your optogenetic experiments.

 

Image
Get a quote

For more background and in-depth information check out our publications area to find some reviews for easy reading and some exciting papers.

We also highly recommend addgene and optobase as resources for your experimental planning.