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

Abstract

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',5'-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 µm²) 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.

Introduction

Optogenetics, born as a tool that revolutionized neurosciences, is now a growing research field and a rising technology routinely used by many laboratories worldwide and across various research areas in biology. We developed bPAC-nLuc, a versatile optogenetic protein, by fusing a light-sensitive adenylyl cyclase (AC) from Beggiatoa sp. (bacterial photoactivatable adenylyl cyclase; bPAC) to nanoluciferase (nLuc)^1,2,3. When stimulated with blue light, bPAC produces the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). nLuc is a recently developed small luciferase that, in the presence of one of its substrates, can generate bioluminescence and activate cAMP production⁴. Thus, this optogenetic protein can be activated transiently by using brief light pulses or steadily with Furimazine or other luciferase substrates, allowing us to mimic different cAMP signaling patterns and assess cellular responses (activation of transcription factors, gene expression, cell proliferation, migration, etc.). Recent advances in 2^nd messenger signaling have emphasized the significance of events occurring in very restricted cytosolic regions (e.g., endosomal cAMP production for cAMP response element-binding protein (CREB) phosphorylation or Ca²⁺ microdomains for nuclear factor of activated T-cells (NFAT) translocation to the nucleus)^5,6. Therefore, developing consistent and systematic strategies to evaluate, mimic, and block signaling from these compartments in live cells is important. To show the ability of bPAC-nLuc to be specifically activated in different cell compartments, we co-transfected a hepatoma-derived cell line (HC-1) with the nuclear-targeted bPAC-nLuc and H208, a Förster resonance energy transfer (FRET) cAMP sensor (NLS-bPAC-nLuc; NLS-H208). HC-1 cells that derive from the HTC line are devoid of assayable AC activity, which results in very low basal cAMP levels, making it ideal to measure putative cAMP production while taking advantage of the full dynamic range of FRET sensors^7,8. Using a solid-state laser (445 nm, LDI-7, 89 North) connected to a point scanning system (UGA-42 Geo, Rapp OptoElectronic), we describe a protocol to systematically stimulate very small circular areas or spots (~1 µm²) within individual cells. The point scanning system was connected to one of the backports of a two-deck microscope, which allowed us to stimulate cells and perform FRET measurements simultaneously via an independent lightpath. We present a method in this protocol where the SysCon Geo software, supplied with the stimulation system by Rapp OptoElectronic, is employed to perform a comprehensive scan of the cytoplasm of cells. The approach involves generating a cAMP response map by setting up a sequence of illuminations that stimulate cells in a grid pattern (Figure 1).

Protocol

1. HC-1 cell culture and preparation for imaging

1. Maintain HC-1 cells in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/L), streptomycin (100 mg/L), and L-glutamine in 10 cm dishes and incubated at 37 °C, 5% CO₂, in 95% humidified air.
2. Passage the cells every 2-4 days once cells are ~90% confluent using 1:5 or 1:10 dilutions.
3. Seed cells for transfection on glass coverslips 2 days before the experiment.
   1. Working under sterile conditions in a tissue culture hood with pre-warmed media and PBS, aspirate the media from a near-confluent dish of cells and gently wash with 5 mL of PBS.
   2. Aspirate the PBS and add 1 mL of trypsin. Incubate at 37 °C for 3-5 min until cells have detached.
   3. Resuspend the cells in 4 mL of media and transfer the cell suspension to a 15 mL conical tube.
   4. Use a hemocytometer to count the number of cells and calculate the cell density.
   5. Dilute the cells to a density of 1.5 x 10⁴ cells/mL.
   6. Pipette 2 mL of the cell suspension onto 25 mm glass coverslips with poly-D-lysine coating in a 6-well dish.
   7. Incubate the cells for approximately 24 h in an incubator until 70% confluence.
4. Transfect the cells using a transfection kit following the manufacturer's instructions using a ratio of 0.25 µg of NLS-bPAC-nLuc DNA/0.75 µg of NLS-H208 DNA per 3 µL of transfection reagent and 2 µL of P3000 reagent diluted in Opti-MEM media lacking phenol red.
5. Place the cells in the incubator for ~48 h to allow for sufficient bPAC-nLuc expression, essential for producing detectable levels of cAMP.
   NOTE: The minimum required level of bPAC-nLuc expression will vary depending on the potency of the stimulation system and the parameters of the stimulation.
6. Prepare cells on coverslips for imaging.
   1. Aspirate the media from one well and wash twice with 2 mL of PBS.
   2. Carefully lift the coverslip using a pair of forceps and place it in the imaging chamber.
   3. Add 1 mL of Opti-MEM lacking phenol red to the imaging chamber. Cells can also be imaged in HBSS or any other physiological fluorescence-compatible solution.

2. Light simulation and live cell imaging

1. Perform cell culture and manipulation of cells expressing NLS-bPAC-nLuc in a dark environment. To avoid exposure to wavelengths <500 nm, use a red safelight lamp (Table of Materials) with a 13 W amber compact fluorescence bulb (Table of Materials).
2. Perform experiments in a motorized two-deck microscope (Table of Materials) equipped with a 6-line multi-LED light engine (Table of Materials), an emission filter wheel (Table of Materials), XY stage (Table of Materials), an ORCA-Fusion Digital CMOS camera (Table of Materials), and a 100x/1.4 NA oil objective (∞/0.17/FN26.5). Capture images using digital microscopy software (Table of Materials).This setup is capable of stimulating cells and performing FRET measurements simultaneously by using an independent lightpath provided with a ZT458rdc dichroic filter (Table of Materials).
   NOTE: Any acquisition setup can be used if it allows simultaneous optogenetic stimulation and FRET/intensiometric imaging with a compatible sensor.
3. Turn on the UGA - 42 Geosystem. Turn on the microscope and set it up with the appropriate parameters to acquire with the H208 FRET sensor. The acquisition configuration will depend on the expression levels of the FRET sensor. The typical parameters are 40 ms simulation time for YFP/mScarletI, 15% LED power, and 0.5 Hz.
4. Open the SysCon software and the imaging software that controls the microscope.
5. On the Camera window, select the Calibration Tab and choose the laser wavelength compatible with the optogenetic protein. To stimulate bPAC-nLuc, use a wavelength of 445 nm.
6. Calibrate the system before each use. This is important because small variations in the setup, or even changes in room temperature (RT), can alter the precision of the stimulation.
7. On the camera window, select the Camera tab and click <Start Acquisition> to start the acquisition from the imagining software.
8. In the Image Viewer window, visualize a live or prior acquisition of the fluorescence of the cells taken from the imaging software, which will guide the positioning of the stimulation objects.
9. Use the Click and Fire mode to quickly evaluate the responsiveness of an individual cell before starting the experiment.
10. Using the Sequence-Manager window, select <add> to create a new sequence.
11. By properly calibrating the system, draw objects of various sizes and shapes in the Image Viewer window that correspond precisely to the stimulation patterns projected onto the cells. This ensures that the patterns accurately match the intended stimulation objects drawn.
12. In the Image Viewer window , select the Round icon in the toolbar on the left to draw circular stimulation objects.
13. Create multiple small identical circles and distribute them evenly to ensure the homogeneous coverage of the entire cytoplasm of the cells to be stimulated.
14. Right-click on each one to verify the homogeneity of the circular stimulation objects. A window will appear; use it to examine their radius, position, etc.
15. Set the radius between 1 and 5, depending on the objective used and the size of the cell.
16. To help with the even spacing and alignment of the circles, use the <Snap to Grid> button in the View subwindow. Additionally, set the properties of the grid by using the <Set Grid> button.
17. Ensure that the grid or matrix of evenly spaced circular stimulation objects fully covers the cell, and its size (e.g., 6 x 6 or 10 x 10) is determined by the size of the cell. As a control for null stimulation that should not trigger any cellular events, include some circles positioned outside the cell.
18. Each object created in the Image Viewer window will appear in the Timeline window.
    1. In the Object Timing subwindow, configure the temporal properties of each stimulation object (start time, duration, delay).
    2. In the Lightsource subwindow, configure the wavelength and intensity of each object. For this protocol, ensure each object uses the same wavelength and is identical in duration and intensity. The start time will be different for each object, and the delay between them can be varied depending on the expected outcome to avoid superimposing the effect of different stimulations.
19. In the Timeline window, alter the sequence in which each stimulation object will be activated if required. Configure stimulations in a regular pattern (e.g., left to right, top to bottom) or in a random pattern, depending on the particular experiment.
20. Upload the sequence to the system and configure the number of sequence cycles (runs) the system will perform.
21. Place the regions of interest (ROIs) in the imaging software at the desired positions.
22. Start acquiring fluorescence images with the microscope and press <Play> at the UGA-42 window.

3. Data analysis

1. Although the software provided by the stimulation system allows saving sequences for further use, it does not record the actual experiment information. Therefore, meticulously document all details related to the stimulation spots and sequences employed during the experiment, including parameters such as position, intensity, duration, number of runs, etc. Document any other relevant experimental conditions, such as pharmacological agents or specific solutions.
2. During the experiment, position the designated ROIs at the imaging software to capture fluorescence intensity changes from the entire cell or specific locations within the cells.
3. Pay special attention to the positioning of the ROIs relative to the stimulation spots. Measure the changes in fluorescence intensity either within or far from the stimulated areas. Adjust the positions of the ROIs post-experiment, providing the imaging software to allow saving images acquired during the experiment and not just the ROI intensity values.

Representative Results

The results presented in Figure 1 show that only stimulations directed to the cell nucleus were able to generate measurable cAMP elevations. This confirms that NLS-bPAC-nLuc is expressed exclusively in the nuclear compartment of HC-1 cells. It is possible to precisely stimulate an optogenetic protein using this grid/matrix pattern to map its intracellular distribution. Additionally, the higher cAMP elevations towards the nuclear center reflect the higher mass of bPAC-nLuc simulated by central spots compared to the ones placed at the nuclear periphery (Figure 1).

Figure 1: The grid stimulation protocol and the cAMP response map. Sequential blue light stimulations (445 nm; 30 s interval between pulses; pulse train: 50 ms, 500 ms total duration, 10 Hz, 7% laser power) on HC-1 cells transfected with NLS-H208 (cAMP sensor) and NLS-bPAC-nLuc. The left panel displays the yellow fluorescent protein (YFP) fluorescence from the H208, which was superimposed with the actual light pulses captured by the sCMOS camera represented in a single image. Traces on the right show transient cAMP increases triggered by NLS-bPAC-nLuc stimulation at each stimulation spot. The background-subtracted NLS-H208 fluorescence was measured using a single ROI covering the entire nucleus, and normalized ratios (R/R0) were calculated. Responses in the outer areas (rows 1 and 7, columns A and G) were negative and are not shown. Bar = 3 µm. Please click here to view a larger version of this figure.

Discussion

The objective of this study was to precisely monitor the intracellular distribution of an optogenetic protein and assess its performance within a particular cytoplasmic compartment. We also showed the precise stimulation capabilities of a point scanning system on cells expressing an optogenetic protein. To achieve this, we employed a nuclear-targeted bPAC-nLuc with high expression levels but a very confined distribution limited to the nucleus. The results showed that stimulation spots separated by only ~1 µm can either trigger cAMP production or not, depending on the cellular distribution of the optogenetic protein; only stimulations directed to the nucleus were able to raise cAMP levels. In this protocol, the goal was not to generate localized cAMP elevations but rather to show that brief light stimulations can be accurate enough to activate optogenetic proteins in very restricted areas of the cell without stimulating the surrounding proteins. However, this approach constitutes a necessary control for the study of other optogenetic proteins with diffuse distributions and/or the generation of localized cAMP elevations. This can be achieved by varying the expression levels of the optogenetic protein, the intensity and frequency of the stimulation, etc. To accurately measure these localized signals, careful consideration of acquisition parameters and sensor characteristics is crucial. This includes selecting appropriate ROI positions and sizes, determining the acquisition frequency, and choosing FRET/intensiometric sensors with optimal affinity, intensity, dynamic range, and dissociation constant.

In a recently published article, we used a similar strategy to functionally demonstrate the distribution of a nuclear-targeted bPAC-nLuc and the capability of a modified phosphodiesterase (PDE; ΔRI-PDE8) to abolish cAMP elevations in a thyroid-derived cell line⁹. In this article, we conducted cell stimulation along a straight line instead of a grid, and the illumination spots were placed relative to cellular landmarks (e.g., nuclear center, nuclear edge, etc.) rather than in a systematic pattern. Remarkably, even stimulations directed to the nuclear edge were insufficient to trigger a detectable cAMP response. We reasoned that this was probably due to stimulating an insufficient mass of NLS-bPAC-nLuc⁹. However, in the present protocol, some stimulations directed to the vicinity of the nuclear edge elicited a detectable cAMP increase. This discrepancy could be attributed to various factors, including the expression levels of the bPAC-nLuc, the characteristics of the stimulation, the morphology of the nucleus, the presence of endogenous PDEs, buffer proteins, etc.

While other targeted or non-targeted optogenetic proteins may exhibit more diffuse distributions, this systematic approach supports drawing meaningful conclusions by carefully setting up experiments and analyzing the data. Furthermore, this approach enables targeting specialized cellular structures (e.g., lamellipodia, dendrites, primary cilia, etc.), facilitating comparisons between responses in the distal and proximal regions of these structures and/or the cell body.

This strategy can also be used to assess the activation of downstream elements in the cAMP pathway, such as Protein Kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC1). Furthermore, since bPAC-nLuc is not influenced by the same modulators as endogenous ACs, which can be affected by local interactions with proteins or other factors, it can be assumed to consistently generate the same amount of cAMP given the same level of expression. By examining the reduction of cAMP elevations or the decrease in cAMP levels after a temporary rise, this approach allows the evaluation of cAMP degradation, diffusion, or the functionality and distribution of endogenous or transfected PDEs. This strategy proves particularly useful for assessing any optogenetic protein's behavior at different cellular locations, helping avoid the bias of arbitrarily placing stimulation spots within the cytoplasm.

It is important to take into account that the size of the stimulation spot and its energy will ultimately depend on the particular characteristics of the setup used (light paths, objectives, scanning system, etc.). To ensure accurate results, the relative expression of the optogenetic protein, the sensitivity of the cells to the wavelength used, and any other parameters should be taken into account when conducting each experiment in each particular system. It is also essential to note that we have not studied the full extent to which the system can be driven in implementing smaller stimulation spots and/or denser grids (i.e., less spaced stimulation spots) to systematically assess smaller cellular structures with increased precision. Additionally, we have tested this strategy using only the UGA-42 Geo point scanning system. This approach may be possible using different systems, provided that they can reproducibly generate evenly spaced localized spots. Each system should be optimized to yield similar results.

Finally, the potential stimulation of FRET sensors by the stimulation system should be carefully evaluated. For example, cyan fluorescent protein (CFP) will certainly be stimulated or even bleached by the 445 nm laser used in this protocol. The use of higher laser potencies (or higher transmittance neutral density (ND) filters) will increase the chances of stimulating the fluorescent sensors and, introducing false information to the experiment and/or generating problems with the data analysis. The normal photobleaching of the FRET/intensiometric sensors should also be carefully evaluated. Finally, since bPAC can be sensitive to ambient light and generate cAMP even in dark conditions (the dark activity of bPAC has been measured as 33 ± 5 pmol/min/mg of protein³), cells should be carefully manipulated after transfecting bPAC-nLUC to avoid light contamination (e.g., at the incubator, before and/or during experiments, etc.). Optionally, cells can be incubated with a PDE inhibitor (e.g., 3-isobutyl-1-methylxanthine; IBMX¹⁰) to determine the baseline activity of bPAC-nLuc and confirm that the lighting conditions used are appropriately dark.

Materials

Name	Company	Catalog Number	Comments
13 W Amber compact fluorescence bulb - Low Blue Lights	Photonic Developments
3-Isobutyl-1-methylxanthine (IBMX)	Sigma	I7018
6-line multi-LED Lumencor Spectra X	Lumencor		6-line multi-LED light engine
Corning - DMEM	Thermo Fischer	MT10013CMEA
Corning - Regular fetal bovine serum	Thermo Fischer	MT35011CV
Cover glasses: circles	Thermo Fischer	12545102P
GBX-2 dark red safelight filter 5.5"	Kodak	1416827	Red safelight lamp
Hanks' balanced salt solution (HBSS) 10x	Thermo Fischer	14185052	Diluted to 1x, adjusted pH
LDI-7	89 North
L-Glutamine	Thermo Fischer	BW17605E
Lipofectamine 3000	Thermo Fischer	L3000001	Transfection kit
Olympus IX83 motorized two-deck microscope	Olympus		Motorized two-deck microscope
Opti-MEM, no phenol red	Thermo Fischer	11058021
ORCA-fusion digital CMOS camera	Hamamatsu	C14440-20UP
Penicillin-streptomycin (10,000 U/mL)	Thermo Fischer	15140122
Phosphate buffered solution (1x)	Lonza	17516F
Prior emission filter wheel and filter sets	Prior Scientific, Inc.		Emission filter wheel
Prior Proscan XY stage	Prior Scientific, Inc.		XY stage
Slidebook 6	Intelligent Imaging Innovations		Digital microscopy software
SysCon software	SysCon Software		Software provided by the stimulation system
UGA-42 Geo	Rapp OptoElectronic
UPlanSApo 100x	Olympus		100x/1.4 NA oil objective (∞/0.17/FN26.5)
ZT458rdc dichroic	Chroma Technology Corp		BS, Wavelength (CWL): 498 nm