Assessment of Mitochondrial Fission/Fusion Dynamics in Kidney Proximal Tubular Cells

In our lab we aim to transplant mitochondria to injured organ to recover them. This is why we're developing new operations protocol to isolate efficient and dynamically active mitochondria. Challenges include tracking transplanted mitochondria without staining while accurately assessing their integration, functionality and dynamic behavior with their new recipient cells.

To begin seed kidney proximal tubular cells in glass bottom culture plates. Incubate the cells overnight to allow for adherence and growth. The next day, add Cisplatin to the experimental plate to a final concentration of two micro molar.

Next, configure the lasers of a confocal microscope to 405 nanometers, 488 nanometers for unconverted Dendra2 excitation and 561 nanometers. Turn on the lasers and allow approximately 10 minutes for stabilization. Place the culture disk in the incubation chamber.

Add water around the chamber and set the temperature to 37 degrees Celsius, ensuring the supply of carbon dioxide is maintained. Allow the incubation chamber to equilibrate before imaging. Visualize the cells using higher magnification and resolution for mitochondrial imaging.

Open the drop down menu in the top left corner and select the FRAP Options. Using LAS X software, define the region of interest where photo bleaching will be performed. Now, apply a high intensity laser pulse to bleach the region of interest by setting FRAP lasers to 405 nanometers at 4.00, 488 nanometers at 0.00.

And 561 nanometers at 0.00. Set the pre-bleach to 5.140 seconds for 59 iterations and define the post-bleach timing. For post-bleach imaging, continue to acquire images at low laser intensity for 10 minutes using FRAP lasers at 405 nanometers at 0.00, 488 nanometers at 0.50.

And 561 nanometers at 0.50. To profile the merged green and red mitochondria, first install the RGB Profiler plug-in. Open ImageJ software, then from the File menu locate and open the image file to analyze.

Select the Color mode Composite, check the Autoscale option and open the video or image stack to analyze. Draw a rectangular area over the image and right click to select Duplicate. Select the Duplicate hyperstack option to duplicate all the image stacks.

Select the image and choose RGB in the Image drop down menu to convert the duplicated stack to RGB format. Duplicate the RGB image and select only one frame. If the mitochondria appear curved, draw a line along their length, then, go to the edit menu, choose Selection and click Straighten.

Now, choose a title for the straightened image and set the line width to 20 pixels. Draw a line along the straightened mitochondrion to align it properly. And open the RGB Profiler plug-in.

Once activated, the plug-in will generate a plot showing red and green fluorescence intensities along the drawn line. The height of each peak will be directly proportional to the fluorescence intensity. After fusion, confirm that the red fluorescence has spread into the adjacent green mitochondrion indicating content mixing.

Verify that the intensity plot now shows overlapping red and green peaks. To assess fusion, select healthy mitochondria and photobleached mitochondria for fusion in live cell imaging. Monitor the change in red to green fluorescence ratio over time to indicate fusion events.

Control cells showed elongated mitochondria with yellow matrix indicating mitochondrial fusion of the red and green matrices. Cisplatin-treated cells exhibited fragmented mitochondria with reduced overlap of red and green signals suggesting impaired fusion. Fluorescence recovery in the photobleached region declined over time in control cells indicating active mitochondrial fusion.

Cells treated with Cisplatin maintained a nearly stable fluorescence ratio indicating impaired mitochondrial fusion. The expression of CLRS-1 was significantly reduced in Cisplatin-treated cells compared to controls. Using cell expression of fluorescence and photo-switchable mitochondria help us track mitochondria and assess their dynamic in recipient cells.

Live cell mitochondria morphology measurement reveals real-time cell health, gene tagging and photo-switchable proteins enhance accuracy and robustness. Future research will explore cellular machinerys, both mitochondric and cytoplasmic that coordinate remodeling to shape tubular, spherical or circular mitochondria.