Optogenetics is the rapidly emerging field in biotechnology and biological sciences that combines the genetic expression of light sensitive molecules and the delivery of light to control cells, populations of cells or animal behavior. Often when we think of optogenetics, the first thought is back to the landmark papers where light was used to depolarize cells and cause action potentials (Zemelman et al. 2002, Boyden et al. 2005). While these types of studies are highly prevalent in neuroscience today, a whole other branch of optogenetics exists that uses light sensitive molecules to modulate some biochemical or second messanger pathway. One area, called OptoXRs, is of great interest as therapeautic targets for a variety of pharmaceutical products as a majority of these act through some type of g-protein coupled receptor (GPCR) pathway.

GPCRs

I have reviewed a paper previously that used an optogenetic approach to GPCR modulation. As a reminder, G-Protein Coupled Receptors (GPCRs) are proteins embedded in the membrane of cells that may be modulated by a variety of endogenous and exogenous compounds and drugs. When something like glutamate binds to its GPCR (for instance, mGluR5) it initiates a biochemical signaling cascade that can result in things like increased production of a protein, or re-alignment of cytoskeletal elements such as actin. These GPCRs have highly diverse and complicated signaling pathways and their function is essential in healthy cells, and a variety of diseases result from errors in GPCR signaling and more than 50% of therapeutic drugs target GPCRs. For a more general look at the role of GPCRs a good place to start would be wikipedia whereas the expert reader may look to a recent article in Annual Review of Pharmacology and Toxicology.

Traditional methods for modulating GPCRs are to apply some drug (activate or inhibit) to cells or tissue and then monitor things like cell growth, protein dynamics, cell excitability, etc. Most of these function on time scales in the seconds to minutes domain. For instance, some GPCRs when activated initiate a sequence of events that change things in the nucleus and alter gene transcription. The ability to interact with GPCRs with more precision in space and time has led to the development of a variety of optogenetic approaches, including those utilized below.

Optogenetics and GPCRs Warning: This is not a full review of their work with analysis of their methods. This is a general overview to stimulate interest in reading the paper. Happy to discuss details in comments below! This work was published in March and comes from the lab of Dr. Gautam and colleagues (Karunarathne et al. 2013) at Washington University School of Medicine in St. Louis. They wanted to develop a method to control activation of GPCRs in specific regions of a cell and therefore created an optogenetic technique for precisely targeting opsins (light sensitive proteins) to areas of a cell. These GPCR-Opsins (non-rhodopsin opsins, meaning that there is no requirement for retinal) are activated with specific wavelengths of light and when activated initiate GPCR signaling. They used a variety of opsins (some natural, some that they created by fusing two together) such as red or blue (bOpsin) and coupled them with GPCRs of all three major types (Gi/o, Gq and Gs). Each of these activates unique pathways in a cell so knowing a bit about the biology of the specific receptors they were then able to use light to activate myriad second messengers and signaling molecules. The opsin-GPCRs they made were targeted genetically.

Having spent time to very precisely target their GPCRs, Gautam and colleagues desired a way to control the spatial pattern of light (see above from Figure 2-A) inputs. Because they didn’t require the speed of a DMD based system, a galvo-based device was used to target light to regions of HeLa cells or hippocampal pyramidal neurons. The earlier figures (1-3) are in HeLa cells, just showing (through confocal and/or multi-point confocal imaging) the basic functionality of their system (characterizing the opsins, showing they can activate GPCR pathways, etc). In figure 4, they characterize a protein they make that is activated at 414nm (blue opsin) but has the internal components of a jellyfish opsin that couples with GPCRs of the Gs type. They show nice controls that using light they can activate a specific pathway (cAMP) and it can be blocked with a well known inhibitor of the pathway (forskolin).

Activation of specific GPCRs lead to neurite outgrowth. Using a galvonometer-based targeted light system they show that activating in distinct locations allows for optically induced neurite extension in individual neurons (see cartoon from Figure 6-E). In figure 6 the put the icing on the cake- they use a dynamic system of activation/deactivation to cause optical reprogramming of lamellipodia in a single cell. The power of the method is that they can very precisely in space and time activate or deactivate a GPCR signaling pathway through non-rhodopsin opsins. Key to their success is the ability to target the light very precisely to various regions of interest.

Summary

Optogenetics presents a novel and powerful toolbox for researchers in cell biology and neuroscience. A lot of work and emphasis has been put into the optogenetic probes that directly modulate ion channels (such as channelrhodopsins). The growing field of non-ion channel modulating optogenetic actuators should be closely monitored by researchers in cell biology, developmental biology and other areas of biotechnology and medical research. We are just at the tip of the iceberg of optogenetic approaches throughout life sciences and it is exciting to consider the potential for these tools within research and eventually in the clinic.

References:

Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K: Millisecond- timescale, genetically targeted optical control of neural activity. Nat Neurosci 2005, 8:1263-8. ( Famous paper in “Optogenetics”)

Karunarathne WK, Giri, L, Kalyanaraman V, Gautam, N. (2013) Optically triggering spatiotemporally confined GPCR activity in a cell and programming neurite initiation and extension. PNAS 2013 110 (17) E1565-E1574

Neuron 3 (33): 15–22. 11779476 Zemelman; Lee GA, Ng M, Miesenböck G. (2002).(33): 15–22. PMID

Commercial disclosure: The paper referenced above uses equipment provided by Andor Technology plc. The views expressed here are my own.