Name: Assessment of Mitochondrial Fission/Fusion Dynamics in Kidney Proximal Tubular Cells (Video) | JoVE Journal
Uploaded: 2025-11-14T14:40:00.000+00:00
Duration: 6 min 14 s
Description: Learn to assess mitochondrial fission and fusion dynamics in kidney proximal tubular cells using confocal microscopy and Dendra2 photo-switching fluorescence. This method enables precise tracking of transplanted mitochondria.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.

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
<ol>
	<li>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.</li>
	<li>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 &#181;g/mL). Grow tubules for 2-3 days until they migrate out of the tubules.</li>
	<li>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.</li>
	<li>Seeding cells for experiments: Seed the cells in glass-bottom culture plates at a density of 1-5 &#215; 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&#39;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.</li>
</ol>
2. Induction of stress using cisplatin
<ol>
	<li>Preparation of cisplatin medium
	<ol>
		<li>Prepare a stock solution of 50 mM of Cisplatin by dissolving in DMSO.</li>
		<li>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.</li>
		<li>Add cisplatin to a final concentration of 2 &#181;M to the experimental plate, to have 0.004% as the final concentration of DMSO. 
		NOTE: Prepare 2 &#181;L of 50 mM cisplatin diluted in 1,998 &#181;L of FBS free DMF-12 media, and add 80 &#181;L to the experimental plate.</li>
		<li>Incubate the cells for 24 h.</li>
	</ol>
	</li>
</ol>
3. Imaging
<ol>
	<li>Using the confocal microscope
	<ol>
		<li>Configure lasers to 405 nm (Dendra2 photo-conversion), 488 nm (unconverted Dendra2 excitation), and 561 nm (photo-converted Dendra2, Alexa 594).</li>
		<li>Turn on lasers and allow time for stabilization (~10 min)</li>
	</ol>
	</li>
	<li>Studying fission and fusion events
	<ol>
		<li>Configure lasers to 405 nm (Dendra2 photo-conversion), 488 nm (unconverted Dendra2 excitation), and 561 nm (photo-converted Dendra2, Alexa 594).</li>
		<li>Place the culture disc in the incubation chamber.</li>
		<li>Add water around the chamber and set the temperature to 37 &#176;C. The supply of CO2 should be maintained.</li>
		<li>Allow the incubation chamber to equilibrate. The temperature change will stress the cells.</li>
		<li>Visualize the cells using higher magnification and resolution for mitochondrial imaging.</li>
		<li>Open the drop-down menu in the top-left corner and select the FRAP options (Figure 2A-D).</li>
		<li>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&#39;t affect the results, as individual mitochondria are analyzed.</li>
		<li>Acquire a series of pre-bleach images at low laser intensity to establish baselinefluorescence intensity.Setlasersto405nmwavelength for photo-switching (0.00), 48&#8239;nm (0.20), and 56&#8239;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.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Profiling the merged red and green mitochondria
	<ol>
		<li>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.</li>
		<li>Open ImageJ (Figure 4A).</li>
		<li>From the File menu, locate and open the file to analyze (Figure 4B).</li>
		<li>Select the color mode composite, check the auto scale option, and open the video to analyze (Figure 4C).</li>
		<li>Select a rectangular area to analyze (Figure 4D).</li>
		<li>Hover near the rectangle area selected, right-click, and select duplicate (Figure 4E).</li>
		<li>Select the duplicate hyper stack option, which duplicates all the stacks of images (Figure 4F).</li>
		<li>To convert the duplicated stack to RGB format, select the image and select the RGB option in the image dropdown (Figure 4G-H).</li>
		<li>The RGB image is duplicated, selecting only one frame (Figure 4I).</li>
		<li>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).</li>
		<li>Choose a title for the image and set the line width to 20 pixels (Figure 5B).</li>
		<li>After straightening the mitochondria, draw a line along the mitochondria of interest to ensure it is in line with a straight line (Figure 5C).</li>
		<li>Open the RGB Profiler plugin (Figure 5D).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Analyze fusion events
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol><li>Liesa, M., Palacin, M., Zorzano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+in+mammalian+health+and+disease%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Mitochondrial dynamics in mammalian health and disease.</a> Physiol Rev. 89 (3), 799-845 (2009).</li>
<li>Chen, Z., Berquez, M., Luciani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria%2C+mitophagy%2C+and+metabolic+disease%3A+towards+assembling+the+puzzle%5BTitle%5D)+AND+%22Cell+Stress%22%5BJournal%5D)">Mitochondria, mitophagy, and metabolic disease: towards assembling the puzzle.</a> Cell Stress. 4 (6), 147-150 (2020).</li>
<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+segmentation+and+function+prediction+in+live-cell+images+with+deep+learning%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Mitochondrial segmentation and function prediction in live-cell images with deep learning.</a> Nat Commun. 16, 743(2025).</li>
<li>Jiao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitocytosis%2C+a+migrasome-mediated+mitochondrial+quality-control+process%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mitocytosis, a migrasome-mediated mitochondrial quality-control process.</a> Cell. 184 (11), 2896-2910.e13 (2021).</li>
<li>Kraus, F., Roy, K., Pucadyil, T. J., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function+and+regulation+of+the+divisome+for+mitochondrial+fission%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Function and regulation of the divisome for mitochondrial fission.</a> Nature. 590 (7844), 57-66 (2021).</li>
<li>He, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GTPBP8+modulates+mitochondrial+fission+through+a+Drp1-dependent+process%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">GTPBP8 modulates mitochondrial fission through a Drp1-dependent process.</a> J Cell Sci. 137 (8), jcs261612(2024).</li>
<li>San-Millán, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+key+role+of+mitochondrial+function+in+health+and+disease%5BTitle%5D)+AND+%22Antioxidants%22%5BJournal%5D)">The key role of mitochondrial function in health and disease.</a> Antioxidants. 12 (4), 782(2023).</li>
<li>Ponte, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drp1-mediated+mitochondrial+fission+regulates+calcium+and+F-actin+dynamics+during+wound+healing%5BTitle%5D)+AND+%22Biol+Open%22%5BJournal%5D)">Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing.</a> Biol Open. 9 (5), bio048629(2020).</li>
<li>Sabouny, R., Shutt, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+mitochondrial+dynamics+in+mtDNA+maintenance%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">The role of mitochondrial dynamics in mtDNA maintenance.</a> J Cell Sci. 134 (24), jcs258944(2021).</li>
<li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+tracking+with+two-photon+phosphorescent+terpyridyl+iridium%28III%29+complexes%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Mitochondrial dynamics tracking with two-photon phosphorescent terpyridyl iridium(III) complexes.</a> Sci Rep. 6, 20887(2016).</li>
<li>Chen, W., Zhao, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+in+health+and+disease%3A+mechanisms+and+potential+targets%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Mitochondrial dynamics in health and disease: mechanisms and potential targets.</a> Signal Transduct Target Ther. 8 (1), 333(2023).</li>
<li>Sabouny, R., Shutt, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reciprocal+regulation+of+mitochondrial+fission+and+fusion%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">Reciprocal regulation of mitochondrial fission and fusion.</a> Trends Biochem Sci. 45 (7), 564-577 (2020).</li>
<li>Jin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+adaptation+in+cancer+drug+resistance%3A+prevalence%2C+mechanisms%2C+and+management%5BTitle%5D)+AND+%22J+Hematol+Oncol%22%5BJournal%5D)">Mitochondrial adaptation in cancer drug resistance: prevalence, mechanisms, and management.</a> J Hematol Oncol. 15 (1), 97(2022).</li>
<li>Wai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+membrane+scaffold+SLP2+anchors+a+proteolytic+hub+in+mitochondria+containing+PARL+and+the+i-AAA+protease+YME1L%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.</a> EMBO Rep. 17 (12), 1844-1856 (2016).</li>
<li>Schiavon, C. R., Shadel, G. S., Manor, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+mitochondrial+mobility+in+Charcot-Marie-Tooth+disease%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Impaired mitochondrial mobility in Charcot-Marie-Tooth disease.</a> Front Cell Dev Biol. 9, 624823(2021).</li>
<li>Sivakumar, A., Shanmugarajan, S., Subbiah, R., Balakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+mitochondrial+PTEN-L+determines+cell+fate+between+apoptosis+and+survival+during+chronic+alcohol+consumption%5BTitle%5D)+AND+%22Apoptosis%22%5BJournal%5D)">Cardiac mitochondrial PTEN-L determines cell fate between apoptosis and survival during chronic alcohol consumption.</a> Apoptosis. 25 (7-8), 590-604 (2020).</li>
<li>Simula, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+metabolism+sustains+CD8%2B+T+cell+migration+for+an+efficient+infiltration+into+solid+tumors%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors.</a> Nat Commun. 15 (1), 2203(2024).</li>
<li>Kopek, B. G., Shtengel, G., Xu, C. S., Clayton, D. A., Hess, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlative+3D+superresolution+fluorescence+and+electron+microscopy+reveal+the+relationship+of+mitochondrial+nucleoids+to+membranes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.</a> Proc Natl Acad Sci U S A. 109 (16), 6136-6141 (2012).</li>
<li>Whelan, D. R., Bell, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image+artifacts+in+single+molecule+localization+microscopy%3A+why+optimization+of+sample+preparation+protocols+matters%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.</a> Sci Rep. 5, 7924(2015).</li>
<li>Palmer, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+microscopy+reveals+the+arrangement+of+inner+membrane+protein+complexes+in+mammalian+mitochondria%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Super-resolution microscopy reveals the arrangement of inner membrane protein complexes in mammalian mitochondria.</a> J Cell Sci. 134 (13), jcs252197(2021).</li>
<li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interactome+of+intact+mitochondria+by+cross-linking+mass+spectrometry+provides+evidence+for+coexisting+respiratory+supercomplexes%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes.</a> Mol Cell Proteomics. 17 (2), 216-232 (2018).</li>
<li>Karbowski, M., Cleland, M. M., Roelofs, B. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKarbowski%2C+M+%22Methods+Enzymol%22">Methods Enzymol</a>. , Elsevier. Netherlands. (2014).</li>
<li>Kleih, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+impact+of+cisplatin+on+mitochondria+induces+ROS+production+that+dictates+cell+fate+of+ovarian+cancer+cells%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells.</a> Cell Death Dis. 10 (11), 851(2019).</li>
<li>Bouchareb, R., Yu, L., Lassen, E., Daehn, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+conditionally+immortalized+mouse+glomerular+endothelial+cells+with+fluorescent+mitochondria%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation of conditionally immortalized mouse glomerular endothelial cells with fluorescent mitochondria.</a> J Vis Exp. (187), e64147(2022).</li>
<li>Chu, C. H., Tseng, W. W., Hsu, C. M., Wei, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image+analysis+of+the+mitochondrial+network+morphology+with+applications+in+cancer+research%5BTitle%5D)+AND+%22Front+Phys%22%5BJournal%5D)">Image analysis of the mitochondrial network morphology with applications in cancer research.</a> Front Phys. 10, 855775(2022).</li>
<li>Montecinos-Franjola, F., Ramachandran, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMontecinos-Franjola%2C+F+%22Methods+Mol+Biol%22">Methods Mol Biol</a>. , Springer, US. (2020).</li>
<li>Gaspar, M. L., Chang, Y. F., Jesch, S. A., Aregullin, M., Henry, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+repressor+Opi1p+and+ER+membrane+protein+Scs2p+facilitates+transit+of+phosphatidic+acid+from+the+ER+to+mitochondria+and+is+essential+for+INO1+gene+expression+in+the+presence+of+choline%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline.</a> J Biol Chem. 292 (45), 18713-18728 (2017).</li>
<li>Karbowski, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKarbowski%2C+M+%22Adv+Exp+Med+Biol%22">Adv Exp Med Biol</a>. , Springer, US. (2010).</li>
<li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+quantitatively+revealed+by+STED+nanoscopy+with+an+enhanced+squaraine+variant+probe%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe.</a> Nat Commun. 11 (1), 3699(2020).</li>
<li>Opstad, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+structured+illumination+microscopy+data+of+mitochondria+and+lysosomes+in+cardiomyoblasts+under+normal+and+galactose-adapted+conditions%5BTitle%5D)+AND+%22Sci+Data%22%5BJournal%5D)">Three-dimensional structured illumination microscopy data of mitochondria and lysosomes in cardiomyoblasts under normal and galactose-adapted conditions.</a> Sci Data. 9 (1), 98(2022).</li>
<li>Kunz, T. C., Götz, R., Gao, S., Sauer, M., Kozjak-Pavlovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+expansion+microscopy+to+visualize+and+characterize+the+morphology+of+mitochondrial+cristae%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Using expansion microscopy to visualize and characterize the morphology of mitochondrial cristae.</a> Front Cell Dev Biol. 8, 617(2020).</li>
<li>Teixeira, P., Galland, R., Chevrollier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+microscopies%2C+technological+breakthrough+to+decipher+mitochondrial+structure+and+dynamic%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Super-resolution microscopies, technological breakthrough to decipher mitochondrial structure and dynamic.</a> Semin Cell Dev Biol. 159 - 160, 38-51 (2024).</li>
<li>Kirchweger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Snapshots+of+mitochondrial+fission+imaged+by+cryo-scanning+transmission+electron+tomography%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Snapshots of mitochondrial fission imaged by cryo-scanning transmission electron tomography.</a> J Cell Sci. 138 (9), jcs263639(2025).</li>
<li>Collins, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+morphology+and+mitophagy+in+heart+diseases%3A+qualitative+and+quantitative+analyses+using+transmission+electron+microscopy%5BTitle%5D)+AND+%22Front+Aging%22%5BJournal%5D)">Mitochondrial morphology and mitophagy in heart diseases: qualitative and quantitative analyses using transmission electron microscopy.</a> Front Aging. 2, 670267(2021).</li>
<li>Marshall, A. G., Damo, S. M., Hinton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+focused+ion+beam+scanning+electron+microscopy%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">Revisiting focused ion beam scanning electron microscopy.</a> Trends Biochem Sci. 48 (6), 585-586 (2023).</li>
<li>Adebayo, M., Singh, S., Singh, A. P., Dasgupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+fusion+and+fission%3A+the+fine-tune+balance+for+cellular+homeostasis%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Mitochondrial fusion and fission: the fine-tune balance for cellular homeostasis.</a> FASEB J. 35 (6), e21620(2021).</li>
<li>Munro, D., Baldy, C., Pamenter, M. E., Treberg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+exceptional+longevity+of+the+naked+mole-rat+may+be+explained+by+mitochondrial+antioxidant+defenses%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">The exceptional longevity of the naked mole-rat may be explained by mitochondrial antioxidant defenses.</a> Aging Cell. 18 (3), e12916(2019).</li>
<li>Varuzhanyan, G., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+during+spermatogenesis%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Mitochondrial dynamics during spermatogenesis.</a> J Cell Sci. 133 (14), jcs235937(2020).</li>
</ol>

The authors declare they have nothing to disclose.

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...

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).

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

assessment of mitochondrial fission/fusion dynamics in kidney proximal tubular cells

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

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.

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...

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

Mitochondria are best recognized for their role in ATP synthesis and serve as key regulators of cellular metabolism. Mitochondrial dynamics comprehend the ...

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.

assessment-mitochondrial-fissionfusion-dynamics-kidney-proximal

Research

JoVE Journal

Biology

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

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

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

In this study, we describe a step-by-step protocol and emphasize the key details for determining morphological characteristics of mitochondria in live cells, including sample preparation, image acquisition, and data analysis. This method is commonly used to examine mitochondrial morphology for studying various conditions.

Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

Here we describe the mitochondrial event localizer (MEL), an ImageJ plugin useful in the quantification of the 3-dimensional changes in mitochondrial fission and fusion activity over time. We also describe an image processing pipeline useful for the cleanup of micrographs prior to analysis in ImageJ.

Understanding the Changes in Mitochondrial Morphology through Dynamic and Three-dimensional Fluorescence Micrographs

Here, we describe an in vitro protocol to model hypoxia/reoxygenation damage in the highly metabolic proximal tubular epithelial cells. This model is designed to induce metabolic stress-induced damage, which can be measured through the expression of proximal tubule damage markers and assessment of mitochondrial respiratory function.

Modeling Hypoxia/Reoxygenation Injury in Proximal Tubular Epithelial Cells

Local injections of a mitochondrial dye label terminal Schwann cell mitochondria in vivo for confocal microscopy. This method utilizes proof of concept demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle to provide high-resolution mitochondrial visualization.

Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Learn to assess mitochondrial fission and fusion dynamics in kidney proximal tubular cells using confocal microscopy and Dendra2 photo-switching fluorescence. This method enables precise tracking of transplanted mitochondria.

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

Explore More Videos