This work was supported by JST, PRESTO (A.I.), Core Research for Evolutional Science and Technology (JPMJCR12W2 (R.K.)), Grant-in-Aid for Scientific Research on Innovative Areas (Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan 26119708 (A.I.) and 16H06480 (R.K.)), Scientific Research (A) (Japan Society for the Promotion of Science (JSPS) 24240049 (R.K.)), and Young Scientists (A) (JSPS 15H05326 (A.I.)), and a Grant-in-Aid for Scientific Research on Innovative Areas &#34;Fluorescence Live imaging&#34; of the MEXT, Japan, and Platform for Dynamic Approaches to Living System from the MEXT, Japan.

1. Generation of Stable Cell Lines by the Tol2 System
<ol>
	<li>Transfect plasmid vectors (Figure 1A) of Tol2-based optogenetic modules together with the transposase (Tol2) expression vector (pCAGGS-mT2TP) into C2C12 cells. In all steps, culture cells with DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37 &#176;C (Table 1), in the presence of 5% CO2, otherwise denoted.
	<ol>
		<li>Count trypsinized cells with a cell-counter, and plate 5 x 104 C2C12 cells per well in a 12-well plate one day before transfection.</li>
		<li>Co-transfect 0.375 &#956;g of Tol2-based optogenetic vectors (pAI177 or pAI218), 0.125 &#956;g of a drug selection vector (pAI170) and 0.5 &#956;g of pCAGGS-mT2TP vector by using lipofection reagent.</li>
		<li>Trypsinize transfected cells, and plate them into 100 mm culture dishes one day after transfection.</li>
		<li>Exchange culture medium for transfected cells to culture medium supplemented with 100 mg/mL hygromycin.</li>
		<li>Culture cells for 3 days to eliminate un-transfected cells. Note that medium change is not necessary.</li>
	</ol>
	</li>
	<li>Trypsinize transfected cells, and purify a population of cells expressing fluorescent proteins for selection. For receiver cells carrying pAI177, set the sorting gate to Green-PE channel for purification of mCherry-positive cell population. For photo-sensitive sender cells carrying pAI218, set the sorting gate to APC channel for purification of iRFP713-positive cell population.</li>
	<li>(optional) Establish a clonal cell population by standard methods, such as limited dilution or single-cell sorting by FACS.</li>
</ol>
2. Evaluation of Photo-sensitivity of Engineered Cells at the Population Level
NOTE: This part describes a protocol to check dynamic induction of Dll1 ligand proteins upon blue-light illumination by biochemical assays, such as qPCR and Western blotting.
<ol>
	<li>Set up two types of incubators with or without light sources for a light-induced condition or a dark condition, respectively. Set-up light-intensity of a blue-LED trans-illuminator, as shown in Figure 1B, by using a light meter. Program timings and duration of light illumination by loading a control-script to a single-board micro-controller that allows programmable schedules for illumination (Figure 1C).</li>
	<li>Count trypsinized cells with a cell-counter, and plate 1.0 x 105 photo-sensitive sender cells carrying pAI218 and pAI170 on 35-mm diameter plastic culture dishes (more than 12 dishes for light condition and 1 dish for dark condition) and set the dishes in separate incubators for dark/light conditions. After setting up dishes, keep doors of the incubators closed until collecting cell lysates.</li>
	<li>One and a half days after plating, start light illumination (cells are expected to be more than 80% confluent). Don&#39;t expose cells to light to avoid undesirable photo-stimulation. 
	NOTE: Schedules for illumination depends on the purpose of experiments. Note that a sustained illumination condition (e.g. 1-min duration and 10 min intervals with 40 &#181;mol/m2/s light-intensity) is sufficient for a simple check of photo-induction, and that strong light illumination is harmful to cells. Thus, ensure that harmless parameters for illumination are selected.</li>
	<li>About 2 days after plating, prepare cell-lysate with 30-min intervals. Move sample dishes from incubators to on-ice, and start preparing cell lysates for further analysis.</li>
</ol>
3. Dynamic Sender-receiver Assay at the Population Level: Real-time Monitoring of Cellular Responses upon Optical Stimulation by PMT
<ol>
	<li>Trypsinize and count the numbers of the sender and the receiver cells with standard methods. Prepare a 1-mL total volume of mixing suspension of 2.5 x 104 receiver and 1.25 x 105 sender cells (1:5 ratio). 
	NOTE: 2:1, 1:1, or 1:2 sender-receiver ratios also work with a total cell number of 1.5 x 105.10</li>
	<li>Plate mixed cells in each well of 24-well black plates with culture medium containing 1 mM luciferin.</li>
	<li>Analyze the cells on plate reader for luciferase assay, and confirm that the output signals are not too high for the recording system. 
	NOTE: If output signals are higher than 1 x 106 photon counts/s, they may be saturated. In that case, reduce the concentration of luciferin to optimal values so that output signals are lower than 1 x 106 photon counts/s.</li>
	<li>Set the plate on the recording system, and start a recording program (Figure 1D). For example, start light illumination at 18 h after setting the recording. 
	NOTE: Temporal filtering, such as detrending signals with moving averaging and Savitzky-Golay filtering, are useful for visualization of wave-forms and peak detections.</li>
</ol>
4. Dynamic Sender-receiver Assay at the Single-cell Level: Real-time Imaging of Single-cell Responses under the Control of Optogenetic Perturbation
<ol>
	<li>Plate 0.5 x 105 receiver and 2.5 x 105 sender cells onto 27-mm-diameter-glass-base 35 mm diameter dishes. Ensure that mixing ratios and a total cell number (a cell density) are correctly adjusted.</li>
	<li>One day after plating, exchange medium with 2 mL of the recording medium (phenol-red free DMEM medium supplemented with 5% FBS, penicillin-streptomycin and 1 mM luciferin), and set the glass-base dish on the microscope. If necessary, wash out debris of dead cells with PBS (-). 
	NOTE: It is preferable to do this step in a dark condition.</li>
	<li>Set up an inverted microscope equipped with an environmental chamber at 37 &#176;C and 5% CO2.</li>
	<li>Set up a cooled CCD camera. Ensure that the temperature of CCD sensor is cooled down to the destination value (typically, -90 &#176;C).</li>
	<li>Set parameters for &#34;Multi-Dimensional Timelapse&#34; window in automatic acquisition software to capture images with 5 min intervals and more than 288 times (more than one overnight recording).
	<ol>
		<li>Open &#34;Multi-Dimensional Timelapse&#34; window, select a luminescence channel and a slow 50 kHz read-out mode from the pull-down menu and set 4 x 4 binning with 4-min exposure. Ensure that the read-out mode is set to the slow 50 kHz mode, which is critical for reducing read-out noises to detect weakly emitting bioluminescent light.</li>
		<li>Open &#34;Multi-Dimensional Timelapse&#34; window, select fluorescence channels and a fast 1 MHz read-out mode from the pull-down menu and set 2 x 2 binning with 400 ms exposure. Ensure that the read-out mode is set to the fast 1 MHz mode to reduce the time required for fluorescence imaging.</li>
		<li>Start time-lapse recording by clicking &#34;Acquire&#34; button.</li>
	</ol>
	</li>
	<li>Extract single-cell traces of luminescence channels from time-lapse movies by image analysis software, as follows. First, make luminescence and fluorescence stack images by importing the image data to image analysis software. For luminescence images, remove hot pixels derived from cosmic rays by comparing the intensities of pixels in temporally adjacent images, replacing the pixel values to the temporal average of the previous and next frames (implemented as &#34;SpikeNoise Filter&#34;), apply these processed images to a spatially smoothing filter, and subtract background pixel values of out-field views from each frame. Then, determine a &#34;region of interest&#34; (ROI) for each cell on a fluorescent image at each time point, apply the ROI to luminescent images, and measure the luminescent intensity of each ROI.</li>
</ol>

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The authors have nothing to disclose.

We showed a method to control gene expression dynamics with a periodicity of 2 to 3 h. This time-scale is much shorter than the ones in other conventional systems, including the Tet-On system and the original LightOn system. Key parameters to reach the ultradian time-scales are half-lives of photo-induced molecular products, mRNAs, and proteins. These kinetic parameters may depend on cell types and species. For tuning the kinetics, replacing Hes1 3&#39;UTR sequences with others is a straight-forward way, because it does ...

Cell-to-cell communications play critical roles in embryonic patterning in developmental processes. In vertebrate embryos, the metameric structures called somites are formed along the anterior-posterior body axis with a precise temporal accuracy under the control of a time-keeping clock, called the segmentation clock1. During this process, a group of presomitic mesoderm (PSM) cells are periodically converted into somites in a synchronous manner. This process involves synchronized oscillatory gene expression and PSM cells that oscillate in phase form the same somites. The period of the oscillatory gene expression is around 2 to 3 h in mice and about 30 min in zebrafish. When dissociated, PSM cells lose the synchrony2,3, but when they are re-aggregated, they can self-organize and recover the population synchrony4, suggesting that cell-cell coupling is a key for the synchronized oscillations.
Extensive efforts revealed that signaling molecules in the Delta-Notch pathway are tightly connected to the synchronized oscillations of the segmentation clock genes. Either pharmacological inhibitors or genetic mutations of Notch signaling desynchronize the population of the oscillators. In zebrafish, mutants of Notch signaling components, such as DeltaC, DeltaD, and Notch1a, display asynchronous oscillations5,6. In chick or mouse embryos, not only the Notch ligand Delta-like1 (Dll1) but also the Notch Modulator Lunatic fringe (Lfng) is required for synchronized oscillations7,8,9. However, it has been difficult to test the functional capability of such molecules for dynamic information transfer from cell to cell, because temporal resolutions of conventional perturbation of gene regulation dynamics were not sufficient to investigate the processes of timescales of 2&#8211;3 h (ultradian rhythms).
We have recently developed an integrated method to control and monitor gene expression patterns in mammalian cells10. This technology enables induction of gene expression pulses by periodic light illumination on ultradian time-scales. This protocol represents the methods to establish photosensitive cell-lines and observe dynamic responses of reporter cells by live-cell luminescence monitoring in the contexts of cell-to-cell communications. This method is applicable to the analysis of many other signaling pathways.

<table><tbody><tr><td>FACS</td><td>Becton, Dickinson and Company</td><td>FACSAriaII SORP</td><td></td></tr><tr><td>Camera</td><td>Andor</td><td>iKon M-934</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX-81 ZDC</td><td></td></tr><tr><td>PMT device</td><td>Churitsu eletric corp.</td><td>CL24B-LIC/B</td><td></td></tr><tr><td>Blue LED illuminator</td><td>OptoCode</td><td>LEDB-SBOXH</td><td></td></tr><tr><td>DMEM</td><td>Nacalai</td><td>08459-35</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Nacalai</td><td>26253-84</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma</td><td>172012</td><td></td></tr><tr><td>KRYSTAL24 (black 24 well plate )</td><td>Hi-tech</td><td>303012</td><td></td></tr><tr><td>D-Luciferin Potassium Salt</td><td>Nacalai</td><td>20028-24</td><td></td></tr><tr><td>Light meter</td><td>LI-COR Biosciences</td><td>LI-250A</td><td></td></tr><tr><td>anti-HA-Peroxidase antibody</td><td>Roche</td><td>clone 3F10</td><td></td></tr><tr><td>anti-Actin-Peroxidase antibody</td><td>Wako</td><td>clone 2F3</td><td></td></tr></tbody></table>

an optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

We adapted the LightOn system11,12, which enables photo-induced gene expression in mammalian cells, to the study of genetic oscillators with 2- to 3-h periodicity. This system comprises of two parts: the photo-inducible transcriptional activator hGAVPO and a UAS-promoter cassette to drive transcription of arbitrary genes of interest. To accelerate the pulsatile kinetics of photo-induced gene expression, the polyA sequence in the U...

Watch this Scientific Journal Video about An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions at JoVE.com

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

an-optogenetic-method-to-control-analyze-gene-expression-patterns

Research

JoVE Journal

Genetics

7.6K Views.  Kyoto University. Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Video: An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Neuroscience

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Biochemistry

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Bioengineering

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic ...

Biology

Optogenetic Control of Enzyme Activity Using Light-Regulated Allosteric Switch

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer. This video highlights important historical discoveries relating to gene expression, including the understanding of how distinct combinations of DNA bases encode the amino acids that make up proteins. Key questions in the field of gene expression research are explored, followed by a discussion of several techniques used to measure gene expression and investigate its regulation. Finally, we look at how scientists are currently using these techniques to study gene expression.

Gene Expression Research: Concepts and Techniques

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Summary

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Chapters in this video

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Optogenetic Signaling Activation in Zebrafish Embryos

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

An Overview of Gene Expression