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.