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

Summary

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.

Abstract

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.

Introduction

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 synchrony^2,3, 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 oscillations^5,6. 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 oscillations^7,8,9. 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 2–3 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.

Protocol

1. Generation of Stable Cell Lines by the Tol2 System

1. 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 °C (Table 1), in the presence of 5% CO₂, otherwise denoted.
   1. 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.
   2. Co-transfect 0.375 μg of Tol2-based optogenetic vectors (pAI177 or pAI218), 0.125 μg of a drug selection vector (pAI170) and 0.5 μg of pCAGGS-mT2TP vector by using lipofection reagent.
   3. Trypsinize transfected cells, and plate them into 100 mm culture dishes one day after transfection.
   4. Exchange culture medium for transfected cells to culture medium supplemented with 100 mg/mL hygromycin.
   5. Culture cells for 3 days to eliminate un-transfected cells. Note that medium change is not necessary.
2. 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.
3. (optional) Establish a clonal cell population by standard methods, such as limited dilution or single-cell sorting by FACS.

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.

1. 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).
2. 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.
3. One and a half days after plating, start light illumination (cells are expected to be more than 80% confluent). Don'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 µmol/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.
4. 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.

3. Dynamic Sender-receiver Assay at the Population Level: Real-time Monitoring of Cellular Responses upon Optical Stimulation by PMT

1. 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
2. Plate mixed cells in each well of 24-well black plates with culture medium containing 1 mM luciferin.
3. 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.
4. 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.

4. Dynamic Sender-receiver Assay at the Single-cell Level: Real-time Imaging of Single-cell Responses under the Control of Optogenetic Perturbation

1. 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.
2. 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.
3. Set up an inverted microscope equipped with an environmental chamber at 37 °C and 5% CO₂.
4. Set up a cooled CCD camera. Ensure that the temperature of CCD sensor is cooled down to the destination value (typically, -90 °C).
5. Set parameters for "Multi-Dimensional Timelapse" window in automatic acquisition software to capture images with 5 min intervals and more than 288 times (more than one overnight recording).
   1. Open "Multi-Dimensional Timelapse" 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.
   2. Open "Multi-Dimensional Timelapse" 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.
   3. Start time-lapse recording by clicking "Acquire" button.
6. 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 "SpikeNoise Filter"), apply these processed images to a spatially smoothing filter, and subtract background pixel values of out-field views from each frame. Then, determine a "region of interest" (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.

Results

We adapted the LightOn system^11,12, 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...

Discussion

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'UTR sequences with others is a straight-forward way, because it does ...

Materials

List of materials used in this article

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