The promise of optogenetics in cell biology: Interrogating molecular circuits in space and time

Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA.
Nature Methods (Impact Factor: 32.07). 01/2011; 8(1):35-8. DOI: 10.1038/nmeth.f.326
Source: PubMed


Optogenetic modules offer cell biologists unprecedented new ways to poke and prod cells. The combination of these precision perturbative tools with observational tools, such as fluorescent proteins, may dramatically accelerate our ability to understand the inner workings of the cell.

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Available from: Orion D Weiner, Oct 06, 2014
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    • "Blue light photoreceptors and photosensory domains derived from plants and fungi have been tested as light-inducible molecular switches or photoswitches to optogenetically control specific molecular processes in living cells, such as cellular signaling processes and gene expression (Fig. 1A) [1]. However, these natural photoswitches suffer from their slow switch-off kinetics [2]–[6] that prevents them from accurately controlling spatiotemporal activities of cellular proteins [7]. If photoswitches with faster switch-off kinetics than natural photoswitches become available through mutagenesis, they should provide a powerful tool for more spatially and temporally confined optical control of protein activities in the cells. "
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    ABSTRACT: Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3×10(3) s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.
    PLoS ONE 12/2013; 8(12):e82693. DOI:10.1371/journal.pone.0082693 · 3.23 Impact Factor
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    • "Among approaches to overcome these limitations, recently developed optogenetic tools are promising. They allow us to rapidly control the amplitude, location and timing of activity of specific signals rapidly [11]. They also diminish the problem of variance among cells, because we can assess the effects of activated signaling on the same cells. "
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    ABSTRACT: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons.
    PLoS ONE 08/2013; 8(8):e70861. DOI:10.1371/journal.pone.0070861 · 3.23 Impact Factor
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    • "In recent years, optogenetic tools have been devised to activate proteins within cells using light [Toettcher et al., 2011]. These techniques make use of conformational changes occurring in proteins after light is absorbed. "
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    ABSTRACT: We have developed an optogenetic technique for the activation of diaphanous-related formins. Our approach is based on fusion of the light-oxygen-voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This "caged" diaphanous auto-regulatory domain was inactive in the dark but in the presence of blue light rapidly activated endogenous diaphanous-related formins. Using an F-actin reporter, we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9-fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous-related formin function in cells. © 2013 Wiley Periodicals, Inc.
    Cytoskeleton 07/2013; 70(7). DOI:10.1002/cm.21115 · 3.12 Impact Factor
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