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-cou...

<ol>
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Commun. 3, 1141 (2012).</li><li>Shimojo, H., Isomura, A., Ohtsuka, T., Kori, H., Miyachi, H., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+control+of+Delta-like1+in+cell+interactions+regulates+dynamic+gene+expression+and+tissue+morphogenesis.">Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis.</a> Genes Dev. 30, 102-116 (2016).</li><li>Isomura, A., Ogushi, F., Kori, H., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+perturbation+and+bioluminescence+imaging+to+analyze+cell-to-cell+transfer+of+oscillatory+information.">Optogenetic perturbation and bioluminescence imaging to analyze cell-to-cell transfer of oscillatory information.</a> Genes Dev. 31, 524-535 (2017).</li><li>Wang, X., Chen, X., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+gene+expression+by+a+light-switchable+transgene+system.">Spatiotemporal control of gene expression by a light-switchable transgene system.</a> Nat. 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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 border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">FACS</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:180px;">FACSAriaII SORP</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Camera</td>
			<td>Andor</td>
			<td>iKon M-934</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>IX-81 ZDC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PMT device</td>
			<td>Churitsu eletric corp.</td>
			<td>CL24B-LIC/B</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Blue LED illuminator</td>
			<td>OptoCode</td>
			<td>LEDB-SBOXH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td>Nacalai</td>
			<td>08459-35&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin-streptomycin</td>
			<td>Nacalai</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal bovine serum</td>
			<td>Sigma</td>
			<td>172012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KRYSTAL24 (black 24 well plate )</td>
			<td>Hi-tech</td>
			<td>303012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">D-Luciferin Potassium Salt</td>
			<td>Nacalai</td>
			<td>20028-24&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Light meter</td>
			<td>LI-COR Biosciences</td>
			<td>LI-250A</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">anti-HA-Peroxidase antibody</td>
			<td>Roche</td>
			<td>clone 3F10</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">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 ...

The overall goal of this experiment is to observe cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This method can help answer key questions of how cells communicate with each other through the Notch signaling pathway, particularly of how cells transmit the oscillatory information to each other. The main advantage of this technique is that we can control and monitor gene expression dynamics with very high precision.Demonstrating the procedure will be Akihiro Isomura, a research fellow from my laboratory who developed this new technology. To transfect plasmid vectors of Tol2-based optogenetic modules together with the transposase expression vector into C2C12 cells, first count trypsinized cells with a cell counter. Plate 50, 000 C2C12 cells per well in a 12-well plate one day before transfection.Then, co-transfect 0.375 micrograms of Tol2-based optogenetic vectors, 0.125 micrograms of a drug selection vector, and 0.5 micrograms of the transposase expression vector by using the lipofection reagent. Trypsinize the transfected cells, and plate them into 100-millimeter culture dishes one day after transfection. Exchange the culture medium for the transfected cells to culture medium supplemented with 100 milligrams per milliliter hygromycin.Culture the cells for three days to eliminate untransfected cells. Note that medium change is not necessary. Next, trypsinize the transfected cells, and purify a population of cells expressing fluorescent proteins for selection by sorting the receiver and photo-sensitive cells, as detailed in the text protocol.Set up two types of incubators with or without light sources for a light-induced condition or a dark condition. Use a light meter to set up the light intensity of a blue-LED transilluminator. Measure the light intensity after closing the door.Program timings and duration of light illumination by loading a control script to a single-board microcontroller that allows programmable schedules for illumination. Count the trypsinized cells with a cell counter, and plate 100, 000 photo-sensitive sender cells carrying pAI218 and pAI170 on 35-millimeter-diameter plastic culture dishes. Set the dishes in separate incubators for dark and light conditions.After setting up the dishes, keep the doors of the incubators closed until collecting the cell lysates. One and a half days after plating, start light illumination. Don't expose cells to uncontrolled light to avoid undesirable photo-stimulation.So, do this step without opening the door. Note that opening the door here is only for demonstration. About two days after plating, move sample dishes from incubators to ice in 30-minute intervals, and start preparation of cell lysates for further analysis.To monitor the cellular responses upon optical stimulation by PMT in real-time, first trypsinize and count the numbers of sender and receiver cells with standard methods. Prepare a one-milliliter total volume suspension of 25, 000 receiver and 125, 000 sender cells. Plate the mixed cells in each well of 24-well black plates with culture medium containing one-millimolar luciferin.Analyze the cells on a plate reader for the luciferase assay to confirm that the output signals are not too high for the recording system. Next, set the plate on the recording system, and start a recording program. For example, start light illumination at 18 hours after setting the recording.For real-time imaging of single-cell responses under the control of optogenetic perturbation, first plate 50, 000 receiver and 250, 000 sender cells onto 27-millimeter-diameter-glass-base 35-millimeter-diameter dishes. Ensure that mixing ratios and total cell number are correctly adjusted. One day after plating, exchange medium with two milliliters of the recording medium.Set the glass-base dish on an inverted microscope equipped with an environmental chamber at 37 degrees Celsius and 5%carbon dioxide. Set up a cooled CCD camera. Ensure that the temperature of the CCD sensor is cooled down to the destination value.Set parameters for multi-dimensional time-lapse window in automatic acquisition software to capture images with five-minute intervals and more than 288 times. In the multi-dimensional time-lapse window, select a luminescence channel. Ensure that the readout mode is set to the slow 50-kilohertz readout mode, which is critical for reducing readout noises to detect weakly emitting bioluminescent light.In the multi-dimensional time-lapse window, select fluorescence channels. Ensure that the readout mode is set to the fast one megahertz mode to reduce the time required for fluorescence imaging. Finally, set two-by-two binning with 400-millisecond exposure.Next, select a journal tab to set up schedules to stimulate the cells with periodic blue-light illumination. Click the plus button to add a new journal setup. Select a journal file from a journal box, choose multiple time points, and set values in initial point and interval boxes to schedule for stimulation.Ensure that the specified journal file includes sequential protocols for selecting illumination, shutter opening, delay, shutter closing, and delay. Next, set up schedules to stimulate cells with periodic blue-light illumination. Finally, start time-lapse recording by clicking the Acquire button.Finally, extract single-cell traces of luminescence channels from time-lapse movies, as described in the text protocol. Representative results of optogenetic sender-receiver assays are shown here. The photo-inducible sender cells produced oscillatory patterns of Delta ligand expression in the presence of periodic blue-light illumination, as expected.When receiver cells are co-cultured with the photo-sensitive sender cells and exposed to repetitive light illumination, a real-time bioluminescence recording system detects cyclic responses of receiver cells in various conditions of mixing ratios. Moreover, time-lapse microscopy with repetitive light illumination also reveals the synchronized responses of receiver cells at the single-cell level. After its development, this technique paved the way for researchers in the field of cell biology to explore how dynamic information of gene expression is transferred in cell-cell interactions.

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7.5K ビュー.  Kyoto University. The overall goal of this experiment is to observe cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This method can help answer key questions of how cells communicate with each other through the Notch signaling pathway, particularly of how cells transmit the oscillatory information to each other. The main advantage of this technique is that we can control and monitor gene expression dynamics with very high precision.Demonstr...