A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

The overall goal of the following experiment is to measure mitochondrial fusion in highly resolved 3D images of multiple cells in parallel. This is achieved by first expressing mitochondrial photo photoable, GFP in the cells of interest. Next, the GFP is photo activated in a small region of the mitochondrial population in each cell.

Then six optical sections of each cell are collected every 15 minutes until signal equilibration is reached in order to assess the extent of mitochondrial fusion, ultimately a dilution of the mitochondria targeted photo activat, GFP intensity in cells undergoing mitochondrial fusion based on quantification of the fluorescent signal can be assessed. The main advantage of this technique is that it is automated and therefore high resolution data of multiple cells can be collected more quickly. Though this method will be demonstrated using INS one cells and provides insight into diabetes, it can be applied to other cell types to study the whole of mitochondrial fusion in aging as well as metabolic and neurodegenerative disease.

Visual demonstration of this method is critical as the imaging automation steps are not described elsewhere. After culturing INS one cells to 80%confluence detach the cultures with 0.05%trypsin and plate the resuspended cells onto top poly de lysine coated cover slip bottomed imaging plates. After two days of culture, add mitochondrial matrix targeted adenoviral photo activate GFP to the plates.

Then after culturing the cells for 24 hours, exchange the media and allow the cells to grow for two more days. On the day of imaging, add seven to 15 nano molar of TMRE to the imaging plates and equilibrate the cultures for at least 45 minutes. During this time, turn on the 5%carbon dioxide, the incubator and the microscope after the microscope has equilibrated.

Under the acquisition tab, click on show manual tools. Open the imaging setup panel and then choose channel mode and switch. Track every frame in the light path panel.

Choose LSM and channel mode. Then from the specimen icon working upward select rear MBS six 90 plus and MBS 4 88 at the bottom of the panel, check the TPMT to enable visualization of the brightfield or DIC channels. Finish adjusting the light path parameters by setting the range for the GFP dye to 490 to 540 nanometers and to 580 to 700 nanometers for the TMRE dye.

Finally, open the acquisition mode and set a 512 by 512 pixel scan field in the frame size, pull down menus and an average of four x in the averaging number menu. Now using an objective lens, focus on the cells with a halogen light. Open the pinhole to maximum and scan to find the bright green photo activat GFP expressing cells.

Choose an arbitrary laser power photo, activate the cell and then take a Z series of optical sections through it to see if the activation is sufficient. Then adjust the imaging 488 nanometer laser to a low power without maximizing the detector gain completely to keep a good signal to noise ratio. After making sure the signal is not saturated, confirm that the photo activat GFP signal is co localized with the TMRE signal.

Finally, open the pinhole and increase the gain on the detector and then find a new photo activat GFP expressing cell and specify the zoom factor. Then set the Z series range to collect six slices and save the imaging method. Now open the multi-time window in the left panel.

Select the saving panel and click on select image folder to specify the location where the files will be saved. Then in the acquisition panel, select the just saved imaging method In the scan configuration pull down menu. Next, select the marked Z middle of Z stack.

Now open the blocks panel and select single block at each location. Then click on add blocks for each interval to be measured, open the timing panel, select weight interval, and then type zero into the weight interval before block at first location only box. After selecting the location panel, choose move focus to load position between locations and the load scan config when move to LO or next LO clicked under the edit locations list, choose clear all and then choose multiple locations motorized stage under the bleach panel, click the bleach box and designate the configuration file in the config pull down menu.

Then in the bleaching window of the main software, save the appropriate photo activation method in the region's panel. Choose the ROI for this particular cell and add this ROI to the multi-time by choosing add current region to ROI list window. Be sure to select the same location in the ROI pull down menu.

Finally, for each of the 10 cells to be followed over time, perform the following sequence. First, when finding a cell indicate its stage position in the location panel and specify its particular scan configuration method in the acquisition panel. Be sure to specify the scan configuration for all blocks.

Then in the main regions panel, choose the region of interest to be photo activated. Next in the bleach panel, save the ROI and load it in the ROI window. Then erase the ROI in the main regions window and reset the scan.

Zoom to one, finally find the next cell and set the zoom. After repeating the process for the next nine cells, check that each stage location and all blocks have the appropriate scan configuration designated and that the first block at each location corresponds to the appropriate photo activation method while the rest contain a mock method. Then select run for cells in which the photo activat GFP signal mostly co localizes with the red TMRE signal.

Subtract the background in the photo activat GFP images, then configure the region measurements to report the average intensity. Then export the data to an Excel file for each slice of the ZS stack. After omitting the planes that do not have a signal, divide the ZS stack intensity of each time point by the initial photo activated intensity and obtain the percentage of the original signal in the INS one cell.

A significant decrease in signal intensity occurs every 15 minutes for an hour until equilibration of the mitochondrial fusion has been reached. As demonstrated in these projected images of six optical sections quantified every 15 minutes during a typical mitochondrial fusion assay. In these assays, a very low concentration of TMRE is used to help target the photo activat GFP photo activation and to monitor cell health.

Note that the cell nearly complete colocalization of the photo activate GFP in green and TMRE signals in red, and that the cos staining of the dyes demonstrates that the mitochondria are active and not depolarized at low millimolar concentrations. Palmitate fragments mitochondria and inhibits mitochondrial fusion. Thus, as seen in these optical sections of a palmitate treated cell, the mitochondria are fragmented and the signal intensity of the matrix targeted photo activat GFP did not change as much as under normal conditions as demonstrated in the previous figure.

Therefore, the dilution of the photo activat GFP signal seen in this figure is likely to be due to mitochondrial fusion rather than fission While setting up this method, keep in mind to choose GFP and TMRE co localizing cells to photo activate enough of the GFP to sustain a signal, but not too much such that you don't miss the mitochondrial fusion events and to collect your entire mitochondrial volume within the optical sections To determine what happens to mitochondrial networks. In the absence of certain regulatory proteins or signaling molecules, this assay can be run in a number of experimental groups in which genes are silenced or the cells are treated with various agents. The ability to quantify mitochondrial fusion and to statistically analyzes parameter allows the study of mitochondrial dynamics in disease states and enables the development of therapeutic treatments.