Cryo-Correlative Light and Electron Microscopy Pipeline for Nanoscale Mapping of Endoplasmic Reticulum–Mitochondria Contact Sites in Hypoxia-Stressed Human Hepatocytes
Keywords:
endoplasmic reticulum–mitochondria contact sites, cryo-correlative light and electron microscopy, cryo-focused ion beam milling, cryo-electron tomography, organelle ultrastructure, hepatocellular hypoxia, mitochondria-associated membranes, nnU-Net segmentation, calcium signaling microdomainsAbstract
Endoplasmic reticulum–mitochondria contact sites (ERMCS) are ultrastructural platforms governing calcium flux, lipid transfer, and mitochondrial dynamics under physiological and pathological conditions. Despite their recognized role in hepatocellular stress responses, precise nanoscale quantification of ERMCS remodeling during hypoxia has remained technically intractable. Here, we present an optimized cryo-correlative light and electron microscopy (cryo-CLEM) workflow integrating fluorescence-guided cryo-focused ion beam milling, dual-axis cryo-electron tomography, and automated segmentation via a retrained nnU-Net convolutional architecture. Applied to HepG2 and primary human hepatocytes exposed to 1% O₂ for 6–48 hours, this pipeline resolved ERMCS geometry with sub-5 nm precision, revealing hypoxia-induced tethering distance compression and a 2.3-fold expansion of contact-site area. Quantitative proteomics of immunopurified ERMCS fractions confirmed concurrent enrichment of VDAC2, MFN2, and IP₃R1. This methodology constitutes a reproducible, broadly transferable framework for organelle-contact-site research in disease-relevant cellular models.
References
Sharma, V. K., Kushwaha, N., Basu, S., Singh, A. K., & Chakraborty, S. (2015). Identification of siRNA generating hot spots in multiple viral suppressors to generate broad-spectrum antiviral resistance in plants. Physiology and Molecular Biology of Plants, 21(1), 9-18.
Sharma, V., Zheng, W., Huang, J., & Cook, D. E. (2020). CRISPR-Cas RNA targeting using transient Cas13a expression in Nicotiana benthamiana. In RNA Abundance Analysis: Methods and Protocols (pp. 1-18). New York, NY: Springer US.
Мустафаева, Г. А., & Шукюрова, З. Ю. (2022). Биология saturnia pyri (LEPIDOPTERA: SATURNIIDAE) на территории Азербайджана. Международный научно-исследовательский журнал, (4-2 (118)), 107-113.
Shukurlu, Y., & Shukurova, Z. (2024). Three-step kinetic model for fisetin dye diffusion into fibroin fibre. Beni-Suef University Journal of Basic and Applied Sciences, 13(1), 72.
Kumar, R., & Shukurova, Z. Y. (2018). Wild silk moths’ conservation status in India. Proc of the GRI of ANAS, 7(1), 122-129.
Shukurova, Z. Y., & Shukurlu, Y. H. (2024). Retraction Note: Non-mulberry silkworm Saturnia Pyri (Denis & Schiffermüller, 1775) and a new perspective source of biomaterials.
Nikolla, M., Mulliri, J., Ribaj, A., & Tema, A. (2023). Measuring the Efficiency of Public Transport Lines in Albania using DEA Model. WSEAS Transactions on Environment and Development, 19, 300-308.
Гусейнова, Н. Т. К., & Мамедова, Р. Ф. К. (2019). Клетка-основа жизни на земле. Universum: химия и биология, (11-1 (65)), 36-41.
Guseinova, N. T., & Mamedova, R. F. (2021). Mechanisms of heredity and the role of genes in the human body. German International Journal of Modern Science, 6, 4-7.
Мамедова, Р. Ф. К., & Бабаев, М. Ш. О. (2020). Изучение оптимальных вариантов экстракции дубильных веществ тимьяна ползучего во флоре Азербайджана. Сельское хозяйство, (1), 15-24.
