Approaches and Application Examples for Studying Mitochondrial Morphology and Function in Caenorhabditis elegans

doi:10.12300/j.issn.1674-5817.2025.119

Abstract

Abstract:

Mitochondria, as the energy metabolism center of cells, have abnormal dynamic morphology and oxidative phosphorylation function, which are directly related to aging and various diseases. Caenorhabditis elegans (C. elegans) is a widely used model animal. This article systematically summarizes the experimental methods for analyzing mitochondrial morphology and function at multiple scales in the laboratory using C. elegans as a model, mainly including: (1) morphological qualitative analysis, which uses a single-blind manual classification method (such as point, rod, and mesh), although it relies on subjective experience, it is easy to operate and suitable for preliminary phenotype screening; (2) morphological quantitative analysis, which relies on the Fiji/ImageJ platform for automated parameter extraction of mitochondria in specific tissues (such as epidermis and body wall muscles), quantifies network connectivity (number of branch points, network length) through skeletonization algorithm, and calculates fragmentation indices (such as area/perimeter ratio, fragment count) with binarization analysis, achieving objective phenotype comparison; (3) high-throughput image processing, which performs batch processing through macro commands, integrates morphological filtering, threshold segmentation, and parameter export workflow, significantly improving research efficiency for large sample sizes; (4) metabolic function monitoring, which uses the Seahorse XF analyzer to measure in vivo respiratory metabolism in C. elegans. By sequential injection of ATP synthase inhibitor N, N'-dicyclohexylcarbodiimide (DCCD), uncoupling agent carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and respiratory chain inhibitor sodium azide (NaN3), the basal oxygen consumption rate (OCR), ATP coupled respiratory efficiency, and maximum respiratory capacity (respiratory potential) are accurately analyzed to reveal energy metabolism patterns. This article integrates a dual dimensional approach of morphology and function, which not only provides empirical techniques for mitochondrial research on C. elegans, but also extends its framework to mammalian cell and organoid models, promoting the basic research and drug development targeting mitochondria.

Key words: Caenorhabditis elegans, Mitochondria, Oxidative respiration, Fiji data processing, Seahorse assay, Aging model

CLC Number:

Q95-33

SONG Mengjiao,SHEN Yidong. Approaches and Application Examples for Studying Mitochondrial Morphology and Function in Caenorhabditis elegans[J]. Laboratory Animal and Comparative Medicine, 2025, 45(6): 726-737. DOI: 10.12300/j.issn.1674-5817.2025.119.

Figures/Tables 8

Figure 1 Introduction to Batch Process operation interface

Figure 2 Preprocessing procedures and annotationsNote： A-D， Preprocessing workflow， target region of interest （ROI） is selected from original image （A）， cropped （B）， subjected to image enhancement （C）， and followed by external background removal （D）. E， Batch-processing macro for steps A-D， with its annotated script.

Figure 3 Classification of mitochondrial morphology in C. elegans muscle cellsNote： A， Representative images of different mitochondrial morphologies in young （day 1） and aged （day 10） C. elegans. B， Qualitative analysis of mitochondrial morphology changes with aging. Green， blue， and pink represent the percentages of C. elegans with tubular， intermediate， and fragmented mitochondria， respectively.

Figure 4 Procedures and annotations of Mitochondria Analyzer pluginNote： A-B， Plugin operation interface. C-E， Schematic of software processing workflow， the pre-processed image （C） is converted into a binarized image （D） using defined block sized and C-value parameters. The binarized image （D） is then skeletonized via the Skeletonize （2D） function to obtain the image （E） . From the binarized image （C）， parameters such as mitochondrial count， area， perimeter， form factor and aspect ratio can be derived. From the skeleton image （E）， data including branch number （Branches）， branch lengths and branch junctions can be extracted. F， batch processing macro for steps C–E， accompanied by explanatory notes.

Figure 5 Procedures and annotations of MiNA - Mitochondrial Network Analysis pluginNote： A-B， Plugin operation interface. C， Schematic diagram of the software processing workflow. The pre-processed image （a） undergoes binarization via the auto thresholding method （Auto threshold-Otsu） to extract mitochondrial contours （b）. Using the parameters set for Ridge Detection in （B） to obtain the skeleton image （c）. Relevant parameters are calculated from both the binarized image and skeleton image， and a contour map （d） is produced. D， Skeletonize algorithm（Skeletonization）. E， Mitochondria Analyzer binarization algorithm.

Figure 6 Age-dependent mitochondrial morphology changes in C. elegansNote： A， Average mitochondrial area， perimeter， and aspect ratio in muscle cells of young adults （day 1） and aged adults （day 10）. B， Branch length， number of network branches， summed branch lengths mean and summed branch lengths median in hypodermal mitochondria of young （day 1） and aged （day 8） C. elegans. A， n=25 （day 1）， n=57 （day 10）； B， n=25 （day 1）， n=43 （day 8）.

Figure 7 Seahorse workflow

Figure 8 Age-dependent mitochondrial function changes in C.elegans （data from published paper of our lab ［20］）Note： A， Schematic of Seahorse analyzer plates. The analyzer plates consist of a sensor cartridge and a cell culture microplate. a and b are side-view and top-view of the sensor cartridge， with the sensor at center and the compound injection ports at the periphery of each well. Same injection port （i.e.， same color port） is selected for adding same compound. Each probe contains 4 injection ports and two sensors measuring oxygen content and solution pH. B， Typical OCR curve phases. C， Comparison of OCR between young and aged C. elegans. D-G， Comparison of basal respiration （D）， ATP-linked respiration （E）， proton leak （F）， and spare respiratory capacity （G） between young and aged C. elegans.

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