Our lab investigates spatial and temporal regulation of cyclic AMP to understand what conditions trigger specific downstream events. To effectively mimic subcellular activation and inhibition of cyclic AMP signaling, we characterize the distribution of an optogenetic tool called bPAC-nanoluciferase. To test the validity of current cyclic AMP signaling models, we've developed tools to evaluate, mimic, and block cyclic AMP signaling from subcellular compartments in living cells.
This protocol can be used to stimulate and evaluate the distribution and function of optogenetic tools in a systematic manner. Well, our goal was to test if focus light process can precisely activate optogenetic proteins in specific cell zones, thus avoiding the impact on surrounding proteins. This method will be vital for studying proteins with diffuse distributions or if the intended effect is to generate localized increases in cyclic AMP or other signaling molecules.
In optogenetic experiments, the typical approach involves stimulating either an entire field of cells or sometimes smaller designated areas. However, the selection of this area and intensity is often arbitrary. Our systematic approach enables investigators to conduct more accurate experiments, helping to mimic patterns of signaling pathways.
For this study, use cells co-transfected with a nuclear-targeted cyclic AMP sensor and an optogenetic nuclear-targeted protein, NLS-bPAC-nanoluciferase. Culture and manipulate the cells expressing the nuclear bPAC in a dark environment. Use a red safelight lamp to avoid exposure to wavelengths less than 500 nanometers.
Perform the imaging experiments in a motorized two-deck microscope equipped with a multi-LED light engine, an emission filter wheel, motorized XY stage, an ORCA-Fusion Digital CMOS camera, and a 100x magnification oil objective with 1.4 numerical aperture. To capture images, use digital microscopy software. This setup is capable of stimulating cells and performing FRET measurements simultaneously by using an independent lightpath equipped with a ZT458rdc dichroic filter.
After turning on the UGA-42 Geosystem and the LDI-7 lasers, switch on the microscope to set it up appropriately for acquiring data with the H208 FRET sensor. Then, sequentially open the imaging software and the SysCon software that controls the microscope. To calibrate the system before each use, navigate to the Camera window and select the Calibration tab.
Set the laser wavelength compatible with the optogenetic protein. Choose 445 nanometers to stimulate bPAC-nanoluciferase. Select the Camera tab and click Start Acquisition to begin the acquisition from the imaging software.
Select the Calibration tab again. Click the Start Calibration button. In the dropdown list that appears, select the proper calibration mode.
Follow the calibration steps indicated by the manufacturer. If needed, change the imaging software settings to perform proper calibration. Ensure to perform the calibration on a cell-free area of the sample.
Next, using the imaging software, acquire one fluorescence image. Fluorescent cells will appear in the Image Viewer window. Position the cell that will be stimulated using the microscope controls XY within the area of influence, preferably at its center.
Before starting the actual experiment, use the Click-And-Fire Mode to quickly evaluate the responsiveness of an individual cell. In the Timeline window, adjust the cell stimulation parameters, including the light source, duration, and intensity of the stimulus. In the Image Viewer window, click approximately on the center of the cell and evaluate the response.
In the Image Viewer window, select the round icon in the toolbar on the left, and in the dropdown menu, click on Filled to draw filled circular stimulation objects. Repeat this step to create multiple identical circles and distribute them evenly, ensuring homogeneous coverage of the entire cytoplasm of the cell to be stimulated. In the Image Viewer window, click the mouse pointer icon on the left, and then right-click one of the stimulation objects to check and edit its properties.
Alternatively, select all the stimulation objects drawn, and then right-click to edit the properties of all the selected stimulation objects together. In the View subwindow, click the Snap to Grid button. This grid can help maintain even spacing and alignment of the circles to form a proper matrix or array.
Additionally, customize the properties of the grid with the Set Grid button. Distribute the stimulation objects evenly with the help of the grid. Add more stimulation objects if necessary to cover the entire cell.
Ensure that all have the same properties. Each stimulation object previously created in the Image Viewer window will be present in the Timeline window, where one can edit a different set of properties, including start time, duration, delay, intensity, and more. To properly visualize the stimulation objects, first expand the Timeline window.
Now, one can see the temporal properties of stimulation objects in the Timeline window. Select all the stimulation objects and edit their delay and duration in the Object Timing subwindow. In the Lightsource subwindow, configure the object wavelengths and intensities.
Ensure identical wavelength and intensity for all. To prevent super imposing the effect of two or more stimulations, set different start times for each object or vary the delay between them depending on the expected outcome. Next, in the Timeline window, change the sequence in which each stimulation object will be activated if required.
Configure the sequence of stimulations in a regular or random pattern depending on the experiment. After uploading the sequence to the system, configure the number of sequence cycles or runs the system will perform in the Sequence subwindow. To measure the fluorescence changes of the stimulated cell, place the regions of interest in the imaging software at the desired positions.
Start acquiring fluorescence images. Finally, press Play at the UGA-42 window to start stimulating the cells. Observe the actual stimulation beams that can be also captured by the camera and their correlation with the stimulation objects.
Note the change in intensity of the green trace only, which shows the increase in the cyclic AMP concentration measured by the sensor. This occurs specifically when the beam reaches a part of the cell with the minimum amount of bPAC-nanoluciferase. The YFP fluorescence of H208 was used to determine the position of the nucleus.
A region of interest covering the entire nucleus was used to measure changes in the FRET ratio of the H208 sensor due to stimulation at the indicated blue spots. This revealed that transient increases in cyclic AMP occurred only when blue light was directed at areas containing the bPAC-nanoluciferase. This confirmed that the nuclear-targeted bPAC-nanoluciferase was expressed exclusively in the nuclear compartment of HC-1 cells.
