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Method Article

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

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DOI:

10.3791/69268

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November 14th, 2025

 ,  ,  ,  , 
1Lewis Katz Medical School at Temple University

In This Article

Summary

This article describes the use of confocal microscopy and ImageJ to assess mitochondrial fission/fusion dynamics via Dendra2 photo-switching fluorescence.

Abstract

Mitochondria are best recognized for their role in ATP synthesis and serve as key regulators of cellular metabolism. Mitochondrial dynamics comprehend the intracellular and intercellular movement of mitochondria, as well as the processes of fission/fusion. These events are fundamental to maintaining mitochondrial function by maintaining cellular homeostasis, morphology, bioenergetics, quality control, and stress responses. On the other hand, dysregulation of mitochondrial dynamics impacts cellular morphology and function. Precise measurement of mitochondria fission/fusion events can be indicative of cellular health. Current methodologies to measure mitochondria dynamics employ advanced imaging like super-resolution microscopy, fluorescence techniques (Fluorescence Recovery After Photobleaching (FRAP)) for connectivity, and optogenetic tools for spatiotemporal control. Quantitative analysis utilizes computational tools to measure parameters like length, number, and branching, which indicate fission/fusion balance. However, these methods require skills and sophisticated instruments. In this article, we describe the use of confocal microscopy combined with free-to-use tools in ImageJ (Fiji) to study fission/fusion events using the photo-switching property of the dendra2 protein, tagged to mitochondrial cytochrome c.

Introduction

Mitochondria is a dynamic organelle playing a vital role in energy production, cell death, and signal transduction1. Mitochondrial morphology refers to the size and shape of mitochondrial compartments, which are highly responsive to intracellular and extracellular conditions and are crucial for maintaining cellular health2. The morphology of mitochondria can provide key insights into their function, however, alterations in mitochondrial shape can impact cellular metabolism and overall health3. Disruptions of mitochondrial morphology are associated with several diseases, including neurodegenerative disorders, cancer, and metabolic diseases4. Mitochondrial morphology is dynamically regulated by fusion and fission/fusion events, which are crucial for maintaining cellular homeostasis and responding to various metabolic requirements5,6.

Mitochondrial dynamics help the organelle respond to different stressors and metabolic requirements7. It involves continuous fission/fusion, and movement of mitochondria within cells or between cells. Mitochondria adapt to cellular energy demands through changes in morphology via fission/fusion events8. These processes determine mitochondrial shape and influence mitochondrial functions such as energy output, reactive oxygen species (ROS) production, and mitochondrial quality control7,9,10. Further, mitochondrial fission/fusion are counterbalancing mechanisms that maintain a functional mitochondrial network11. Fusion allows for the exchange of mitochondrial contents, promoting complementation and buffering against local damage, while fission enables the segregation and removal of damaged mitochondria via mitophagy7,12. Further, fission helps to segregate damaged components of mitochondria, which are then removed by mitophagy, a selective autophagy process13. Fusion, on the other hand, allows for the complementation of mitochondrial contents, diluting any damaged components14. The key players in mitochondrial Fission/Fusion are Mitofusins (MFN1/2), essential for outer mitochondrial membrane fusion15,16, (OPA1): this dynamin-like GTPase is responsible for inner mitochondrial membrane fusion16. Fission proteins involve dynamin-related protein 1 (Drp1), pro-fission protein13,17. Mitochondrial fission 1 protein (Fis1), Mitochondrial fission factor (Mff), and Mitochondrial Fission Regulator1 Like (MTFR1L). Several techniques, like super-resolution EM, have been used to study mitochondrial ultrastructure18. Single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) have been used to visualize individual mitochondrial mRNA and associated proteins18. Single molecule localization microscopy (SMLM) techniques track changes in membrane spacing19. Stochastic optical reconstruction microscopy (STORM) super-resolution microscopy allows the study of mitochondrial inner membrane complexes20. However, the use of this advanced equipment is not easily accessible. Confocal microscopy is accessible and requires less expertise. Further, the development of mitochondrial-targeted fluorescent proteins has significantly advanced the study of mitochondrial dynamics in living cells21, as we are using photoactivable Green Fluorescent Protein (GFP) to visualize and quantify the mitochondrial fusion22.

To study mitochondrial fission and fusion, the cells are stressed or exposed to drugs like cisplatin, which binds to DNA and induces DNA-damage, culminating in mitochondria-mediated apoptosis23. In this study, we have used photo-switchable tagged Dendra2 mitochondria from photo-activatable mitochondria mice to study the fission and fusion events in cisplatin-treated kidney tubular cells, as they are rich in mitochondria and have a high capacity for regeneration. However, this protocol can be used in any cell type that has photo-switchable tagged Dendra2 mitochondria (Figure 1A-C).

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Protocol

All animal work was performed in accordance with protocols approved by Temple University Institutional Animal Care and Use Committee, Temple University (Animal Protocol-5183). The Pham mice, which have mitochondria expressing Dendra2 protein, were purchased.

1. Preparation of cell culture medium

  1. Isolate kidney proximal tubular cells according to the protocol after they are separated from glomerular endothelial cells using beads24 (Figure 1A). Bouchareb et al., developed a method to isolate and culture glomerular endothelial cells (GECs) from the kidneys of transgenic mice that express fluorescent mitochondria (mito-Dendra2) using magnetic beads. After isolation of GEC, the tubules that are left in the pellet are isolated and cultured.
  2. Culture kidney proximal tubular cells in a 10 cm dish with 10 mL of DMF-12 supplemented with 5% FBS (heat-inactivated), penicillin (100 U/mL), and streptomycin (100 µg/mL). Grow tubules for 2-3 days until they migrate out of the tubules.
  3. Select tubular cells by morphology after starving (with DMF-12 without FBS) for 2 days.
    NOTE: For the experiments, use a disc with 50-70% confluent cells. Count the cells with a hemocytometer before seeding.
  4. Seeding cells for experiments: Seed the cells in glass-bottom culture plates at a density of 1-5 × 105 cells per well, depending on the experimental requirements. Allow the cells to adhere and grow overnight before the addition of cisplatin.
    NOTE: Cisplatin is a hazardous chemotherapy drug, it's crucial to follow safety protocols to protect the personnel and the environment. For this purpose, follow the class II biological safety cabinet, personal protective equipment (PPE), decontamination, spill control, and waste disposal (hazardous drug waste-chemotherapy) instructions: wear double-gloving, googles, decontaminate the work surface, and keep the spill kit near the workstation, clearly label the waste container. For DMSO, wear PPE, work in a fume hood, and follow the spill decontamination and disposal procedures.

2. Induction of stress using cisplatin

  1. Preparation of cisplatin medium
    1. Prepare a stock solution of 50 mM of Cisplatin by dissolving in DMSO.
    2. Plate 1-5 x 106 proximal tubular cells on the glass bottom culture plate, add 2 mL of DMF-12 media.
      NOTE: Any glass bottom culture disc may be used.
    3. Add cisplatin to a final concentration of 2 µM to the experimental plate, to have 0.004% as the final concentration of DMSO.
      NOTE: Prepare 2 µL of 50 mM cisplatin diluted in 1,998 µL of FBS free DMF-12 media, and add 80 µL to the experimental plate.
    4. Incubate the cells for 24 h.

3. Imaging

  1. Using the confocal microscope
    1. Configure lasers to 405 nm (Dendra2 photo-conversion), 488 nm (unconverted Dendra2 excitation), and 561 nm (photo-converted Dendra2, Alexa 594).
    2. Turn on lasers and allow time for stabilization (~10 min)
  2. Studying fission and fusion events
    1. Configure lasers to 405 nm (Dendra2 photo-conversion), 488 nm (unconverted Dendra2 excitation), and 561 nm (photo-converted Dendra2, Alexa 594).
    2. Place the culture disc in the incubation chamber.
    3. Add water around the chamber and set the temperature to 37 °C. The supply of CO2 should be maintained.
    4. Allow the incubation chamber to equilibrate. The temperature change will stress the cells.
    5. Visualize the cells using higher magnification and resolution for mitochondrial imaging.
    6. Open the drop-down menu in the top-left corner and select the FRAP options (Figure 2A-D).
    7. Define the region of interest (ROI) using LAS X software, where photobleaching will be performed.
      NOTE: The region of interest can be drawn at any size, which doesn't affect the results, as individual mitochondria are analyzed.
    8. Acquire a series of pre-bleach images at low laser intensity to establish baselinefluorescence intensity.Setlasersto405nmwavelength for photo-switching (0.00), 48 nm (0.20), and 56 nm (0.20), ensuring the cells are focused.
      NOTE: The values in parentheses following the laser wavelengths indicate laser intensities that can be set to any value since this does not affect photobleaching.
    9. To photo-bleach the cells, apply a high-intensity laser pulse to bleach in the ROI. Set the FRAP lasers to 405 nm (4.00), 488 nm (0.00), and 561 nm (0.00). Set the pre-bleach to 5.140 s for 59 iterations, and post-bleach to the desired timing (Figure 2B) (Prebleach and post-bleach laser intensity are mentioned in brackets).
    10. For post-bleach imaging, continue to acquire images at a low laser intensity to observe the recovery of fluorescence for 10 min. Keep FRAP lasers at 405 nm (0.00), 488 nm (0.50), and 561 nm (0.50).
      NOTE: The bleaching time might differ depending on cell types, in our hands, similar bleaching timings were used for mitochondria in tubular cells and Valve Interstitial Cells (VIC). Once FRAP is concluded, the images or live cell imaging (Figure 3 and Supplementary Video 1) can be done, besides acquiring the images post-bleaching.

4. Image analysis

NOTE: Since mitochondria are tagged with the Dendra2 protein, upon undergoing photobleaching, an irreversible conformational change in the dendra2 protein leads to an increase in the red fluorescence detected through the 561 nm laser. When the red mitochondria fuse with the unbleached green mitochondria, the combination gives a yellow color. Taking advantage of this phenomenon, the dynamic fission/fusion events can be measured.

  1. Profiling the merged red and green mitochondria
    1. Install the RGB profiler plugin to profile the red and green signal across the selection.
      NOTE: Follow the instructions on the website to add the plugin to the Fiji section.
    2. Open ImageJ (Figure 4A).
    3. From the File menu, locate and open the file to analyze (Figure 4B).
    4. Select the color mode composite, check the auto scale option, and open the video to analyze (Figure 4C).
    5. Select a rectangular area to analyze (Figure 4D).
    6. Hover near the rectangle area selected, right-click, and select duplicate (Figure 4E).
    7. Select the duplicate hyper stack option, which duplicates all the stacks of images (Figure 4F).
    8. To convert the duplicated stack to RGB format, select the image and select the RGB option in the image dropdown (Figure 4G-H).
    9. The RGB image is duplicated, selecting only one frame (Figure 4I).
    10. If the mitochondria in the image are curved, draw a line along their length. Then, go to the Edit menu, choose Selection, and click on Straighten (Figure 5A).
    11. Choose a title for the image and set the line width to 20 pixels (Figure 5B).
    12. After straightening the mitochondria, draw a line along the mitochondria of interest to ensure it is in line with a straight line (Figure 5C).
    13. Open the RGB Profiler plugin (Figure 5D).
    14. Once activated, the plugin will generate a plot showing red and green fluorescence intensities along the drawn line. The height of each peak is directly proportional to the fluorescence intensity (Figure 5E).
      NOTE: Mitochondrial morphology can also be calculated using the mitochondria analyzer plugin25. Select the regions used for exposure, analyze the threshold in the selected regions, and then measure the mitochondria morphology and 2D analysis. The area perimeter and other parameters can be selected.

5. Analyze fusion events

  1. Establish pre-fusion baseline: Before mitochondrial fusion, confirm that the red fluorescence (561 nm) is localized to one mitochondrion and the green fluorescence (488 nm) to an adjacent one. The corresponding intensity plot should display two distinct peaks, one red and one green, with minimal overlap.
  2. Confirm post-fusion event: After fusion, the red signal (representing Dendra2 matrix content) should spread into the adjacent green mitochondrion, indicating content mixing. The intensity plot will now show overlapping red and green peaks, confirming a fusion event.
  3. The assessment of fusion events can be determined by carefully selecting the healthy mitochondria and the photobleached mitochondria for fusion in the live cell imaging. A change in the ratio of red/green over time would indicate the fusion event.

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Results

Primary tubular epithelial cells were isolated from the mouse kidney by enzymatic digestion and cultured in DMF-12 supplemented with 5% FBS, 1% penicillin-streptomycin at 37 °C under 5% CO2. Upon reaching 80% confluency, cells were trypsinized (0.25% trypsin-EDTA) and seeded at a density of 5 × 104 cells per well into glass bottom culture plates, pre-coated with collagen.

The culture medium contained cisplatin at a final concentration of µM in DMF-12 supplemented with 0.0...

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Discussion

Mitochondrial fission/fusion are crucial dynamic processes that maintain mitochondrial homeostasis and cellular viability26. Proper assessment of these processes requires advanced imaging techniques to visualize and quantify mitochondrial morphology, dynamics, and function27,28. Various techniques have been used for measuring mitochondrial fission/fusion. Some of the sophisticated tools include Stimulated Emission Depletion (STED) nanoscop...

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Disclosures

The authors declare they have nothing to disclose.

Acknowledgements

This work was supported by research grants from the National Institute of Health grant number 5R01DK135470-03, ISAC-NIH grant 5U24DK128851-03, and American Heart Association postdoctoral fellowship.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Cell Culture 
DMEM/F12 Basal medium (Gibco/Cat. No.11320033 ) Proximal Tubular cell culture 
Fetal Bovine Serum (FBS) 5-10% supplementation (Biowest/Cat. No.S181B) Culture medium enrichment 
Penicillin-Streptomycin 100X (Gibco/Cat. No.15140122) Antibiotic protection 
Collagen IV coating ( Sigma-C5533-5M) Ibidi plate coating 
Trypsin-EDTA 0.25% solution (Gibco/Cat. No.25200056) Cell detachment 
Phosphate-Buffered Saline (PBS) (Gibco/Cat. No.10010023 ) Cell washing 
Treatments 
Cisplatin 2 µM working concentration (Selleck Chemicals/Cat. No.S1166) Stress induction  
Dimethyl Sulfoxide (DMSO) vehicle control (Fisher Scientific/Cat. No.D1391) Cisplatin solvent control 
Imaging 
Phenol Red-Free Medium Live imaging (Gibco/Cat. No.21041025) Reduced background fluorescence 
Molecular Biology 
TRIzol® RNA extraction (Gibco/Cat. No.15596018) Total RNA isolation 
SYBR Green Master Mix qPCR chemistry (Thermo Scientific/Cat. No.K0221) Mitochondrial dynamics gene expression (DNM1L, FIS1, MFN1, MFN2, OPA1) 
Fluorescent Tools 
Dendra2 Mitochondrial matrix tag Jacson Laboratory(PhAMexcised) Photo-switchable reporter (405nm conversion, 488/561nm imaging) 
Microscopes 
Confocal Microscope Leica DMi8 Live-cell imaging and FRAP 405nm/488nm/561nm lasers 
Image J https://imagej.net/software/fiji/downloads 
RGB Profiler Plugin https://imagej.net/ij/plugins/rgb-profiler.html. Fusion event analysis (red/green signal overlap) 

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Mitochondrial DynamicsMitochondrial FusionConfocal MicroscopyFRAP TechniqueDendra2 ProteinImageJ AnalysisLive Cell ImagingMitochondrial Morphology
Assessment of Mitochondrial Fission/Fusion Dynamics in Kidney Proximal Tubular Cells
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