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

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....

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 tr....

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.......

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 function^27,28. Various techniques have been used for measuring mitochondrial fission/fusion. Some of the sophisticated tools include Stimulated Emission Depletion (STED) nanoscop.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)