Optogenetic Approaches: How does Optogenetics work?

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.