At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior

Abstract

Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.

Keywords: illumination; photostimulation; photoswitching; signal transduction; spatiotemporal.

Figure 1

User-defined control of protein behavior using optogenetics. (a) Light-activated channelrhodopsins (blue, right) mimic neurotransmitter-activated ion channel opening (violet, left). (b) Experimental workflow of a typical optogenetic experiment. Optogenetic constructs are stably expressed in a cell (left); user-defined illumination patterns activate optogenetic proteins (middle); cell output, such as an action potential, is measured (right). (c) Biological applications of optogenetic methods. Temporally and/or spatially varying input signals (left) are integrated by a cell (middle) to control and study a variety of cell behaviors (right).

Figure 2

Representative schematics of the five major classes of optogenetic systems. Protein size indicated by relative size of bars, and sample proteins A/B are drawn to a scale of 500 amino acids. (a) Light-induced ion channel (blue) opens in response to blue light to allow cation passage (yellow balls). (b) Jα helix unfolds from AsLOV2 core (green) to uncage a fused protein (gray). (c) Reversible interaction between Phy (red) and Pif (orange) leads to dimerization of attached domains (gray). (d) Light-induced uncaging of affinity domains [positive domain of pMag (dark blue) and negative domain of nMag (pink)] results in dimerization of attached domains (gray). (e) Cry2 (blue) clusters an attached protein (gray) in response to light.

Figure 3

Memory erasure with photoactivatable Rac1 targeted to activated synapses (AS-PaRac1). (a) Schematic of AS-PaRac1 activation in response to a light stimulus. (b) AS-PaRac1 engineered to target active dendritic spines, where blue light stimulation triggers dendritic spine shrinkage and disruption of synaptic connection (red arrows). (c) Repeated motor tasks activate similar synaptic patterns. Expression map of AS-PaRac1 within the adult brain (left) after a series of rotarod trainings and (right) after memory erasure and retraining shows a large amount of overlap in AS-PaRac1 location. Adapted with permission from Reference 97, Macmillan Publishers Ltd., Nature ©2015.

Figure 4

Optogenetic clustering of LRP6c induces Wnt/β-catenin pathway activation in neural stem cells. (a) Schematic of LRP6c oligomerization with Cry2 to induce the Wnt/β-catenin signaling pathway. (b) Live neural stem cell (NSC) fluorescence imaging of Cry2-mCherry-LRP6c upon blue light illumination for 2.5 min. (c) HEK 293 T cells expressing Cry2-LRP6c and a firefly luciferase reporter for β-catenin show Wnt/β-catenin pathway activation upon illumination. Activation is attenuated by constitutively active (CA) β-catenin pathway inhibitor GSK-3β and is comparable to β-catenin pathway activation with the small-molecule agonist CHIR99021. (d) Illumination of NSCs expressing Cry2-LRP6c and a luciferase β-catenin reporter shows increased reporter expression with increasing light dosages (pulse interval defined as time light-off between 500 ms light-on pulses). Wnt3a protein as positive control for Wnt/β-catenin pathway activation. Adapted with permission from Reference 62, Macmillan Publishers Ltd., Nature Methods, © 2013.

Figure 5

In vitro and in vivo technology for optogenetic stimulation. (a) Schematic of a cell culture plate illuminated with a light-emitting diode (LED) array. (b) Programmable LED array for multiwell cell culture stimulation. (c) Image of a digital micromirror device displaying the UC Berkeley logo. A user-defined pattern can be projected onto a sample and either magnified onto a cell culture plate for large-scale patterning or demagnified for subcellular protein control. (d) Image of assembled wireless illumination device (left) and schematic of device implantation (right) by epidural insertion into mouse spinal cord. Adapted with permission from Reference 131, Macmillan Publishers Ltd., Nature Biotechnology, © 2015. (e) Diagram and image of wireless illumination device (left) and implanted device (right) into live mouse for stimulation of peripheral nerve endings around the rear heel, powered by a resonant radio-frequency cavity below. Adapted with permission from Reference 132 Macmillan Publishers Ltd., Nature Methods, © 2015.