Optogenetic control of cell function using engineered photoreceptors

Abstract

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

Figure 1. Mechanism of phytochrome B photoactivation

In dark or far-red light, phyB exists predominantly in a red light responsive ‘Pr’ form. Upon red light stimulation, the covalently bound chromophore phytochromobilin (top) undergoes a reversible Z-E isomerization, resulting in a conformational change in phyB to the Pfr signaling state. This state can be switched back to the Pr state by far-red light illumination. Light dependent effector proteins, such as PIF3 or PIF6, bind the photoreceptor preferentially in one conformational state.

Figure 2. Strategies for engineered optogenetic regulation

a) Allosteric regulation. Photoreceptors or domains such as AsLOV2-Jα that undergo a large structural change upon light binding can be attached to or inserted within other proteins to control enzyme activity or binding interactions. b) Optical dimerizers. Modular light-interacting domains are used to control interactions and localization of fused target proteins (e.g. phyB and PIF3).

Figure 3. Basic schemes for control of cell function by optical dimerizers

a) Split protein reconstitution. Two non-functional parts of a protein (blue and purple half-circles) are brought together by optical dimerizers to restore protein activity in a light-dependent manner. b) Subcellular recruitment. A protein is recruited via dimerizers to a region of the cell where it is active. In the absence of recruitment the protein is nonfunctional. c) Sequestration. A protein is sequestered in an inactive state by a dimerizer, then released to sites of action with light.

Figure 4. Use of optogenetic systems for fine spatial control of cell function

a) Focal activation of Rac-mediated cell protrusion with the phyB/PIF6 system. The catalytic domain of the Rac GEF Tiam (Tiam-DH-PH) is recruited to the plasma membrane via the phyB/PIF6 interaction, where it acts through Rac1 to form lamellipodia. Localized lamellipodia formation in NIH3T3 cells was induced by globally irradiating the cell with infrared (750 nm) light and spot illumination with a red (650 nm) laser. Scale bar 20 μm. Figure adapted with permission from Macmillan Publishers Ltd.: Nature (Levskaya et al., 2009), copyright 2009. b) Local recruitment of phosphatidylinositol 5′-phosphatase (5-ptase) to the plasma membrane using the CRY2/CIBN system. Upon focal light stimulation within COS-7 cells, cytosolic mCh-CRY2-5-ptase is recruited to a nearby region of the plasma membrane via interaction with CIBN. At the plasma memrane, the phosphatase depletes PI(4,5)P2 locally, visualized using the PI(4,5)P2 biosensor iRFP-PHPLCδ1. Images at right were taken of COS-7 cells expressing the constructs before and 10s after delivery of a 100-ms blue-light pulse (location marked by blue square). Scale bar: 5 μm. Figure reprinted with permission from The National Academy of Sciences, Proceedings of the National Academy of Sciences, USA (Idevall-Hagren et al., 2012), copyright 2012. c) Light-directed polarized yeast growth. Yeast cells expressing Cdc24-ePDZb1 and membrane localized Mid2-GFP-LOVpep were exposed to mating pheromone to induce cell cycle arrest. After 30 min, Cdc24-ePDZb1 was spot recruited using a blue laser and cells were imaged after two hours. Figure adapted with permission from Macmillan Publishers Ltd., Nature Methods (Strickland et al., 2012), copyright 2012.

Figure 5. Integrating multiple optical tools for complex control

In this hypothetical scheme, the phyB/PIF optical dimerizer system is combined with a CRY1/phyB system that is light-dissociated. Thus, in blue light, none of the proteins interact. In dark or far-red light, phyB interacts with CRY1, while in red light phyB interacts with PIF. Thus, a protein of interest tethered to phyB can be shuttled from one location to the other (for example, plasma membrane vs. nuclear membrane) via changes in light wavelength.