Lysosomes Keep Mitochondria Healthy [Mar. 18, 2025] DOJINDO LABORATORIES

Lysosomes Keep Mitochondria Healthy [Mar. 18, 2025]

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Lysosomes play a critical role in supporting mitochondria and maintaining mitochondrial homeostasis. This Science Note introduces the latest findings on the intimate relationship between lysosomes and mitochondria, including the mechanism by which lysosomes partially remove mitochondrial components, the process of mRNA transport in neuronal axons, and the compensatory mechanisms that occur in response to lysosomal dysfunction.

Lysosomes drive the piecemeal removal of mitochondrial inner membrane (Nature, 2024)

Lysosomes maintain mitochondrial quality control not only by degrading the entire mitochondria but also by selectively removing damaged sections of the inner mitochondrial membrane (IMM). The damaged IMM protrudes through pores in the outer membrane formed by VDAC1, and nearby lysosomes engulf this IMM portion, leading to the formation of vesicles in the cytoplasm.

Highlighted technique: This study uses a combination of super-resolution microscopy, live-cell imaging, and specific fluorescent probes and proteins to visualize and track in real time the dynamic process of IMM protruding through the OMM and their subsequent uptake by lysosomes.

Related technique Lysosomal function, Mitophagy detection

Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration (Nature, 2024)

Neurons have long axons and require local protein synthesis, which relies on lysosome-related vesicles to transport mRNA within the axon. Disruption of this transport leads to mitochondrial dysfunction and axonal degeneration, suggesting that abnormalities in mRNA transport by lysosome-related vesicles may trigger neurodegenerative diseases.

Highlighted technique: In in vitro experiments with neuronal cells, it is critical to accurately mimic real neurons. While neuronal cell lines often differ in morphology from neurons in vivo, this study uses iPS-derived or primary neurons and employs a microfluidic device to more accurately replicate axons and assess intracellular dynamics.

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired (Nature Communications, 2023)

Dysfunctional mitochondria are normally removed by lysosomes, but when lysosomal function is impaired, an alternative compensatory mechanism facilitates the secretion of accumulated mitochondria into extracellular vesicles (EVs). These EVs containing mitochondria are taken up by macrophages and degraded without triggering inflammation.

Highlighted technique: CD81 and CD63 are markers for extracellular vesicles (EVs) and are key molecules widely used for tracking, quantifying, identifying, and purifying EVs within cells. In addition, membrane staining dyes are often used in experiments to track the uptake of purified EVs by cells or organisms.

Related technique Oxygen consumption rate assay, Exosome staining

Related Techniques (click to open/close)

Target	Kit & Probes
Lysosomal function	Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
Mitophagy detection	Mitophagy Detection Kit
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
First-time autophagy research	Autophagic Flux Assay Kit
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Extracellular ATP detection	Extracellular ATP Assay Kit-Luminescence

Application Note (click to open/close)
> Induction of Mitophagy in Parkin Expressed HeLa cells

Induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial-uncoupling reagent with Parkin expressed HeLa cells

HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37^oC overnight in a 5%-CO₂ incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent, and incubated at 37^oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37^oC for 30 minutes with 250 μl of 100 nmol/l Mtphagy Dye working solution containing 100 nmol/l MitoBright LT Deep Red. After the washing of the cells with Hanks’ HEPES buffer twice, the culture medium containing 10 μmol/l CCCP was added to the well. After 24 hours incubation, mitophagy was observed by a fluorescence microscopy. After removing the supernatant, 250 μl of 1 μmol/l Lyso Dye working solution were added to the cells and incubated at 37^oC for 30 minutes. The cells were washed with Hanks’ HEPES buffer twice and then co-localization of Mtphagy, Lyso Dye and MitoBright Deep Red was observed by confocal fluorescence microscopy.

> Effect of Bafilomycin A1 on Lysosomal Mass and pH
HeLa cells were used to detect changes in lysosomal mass and pH when treated with the lysosomal acidification inhibitor Bafilomycin A1 (Baf. A1). The fluorescence of LysoPrime Deep Red has no change regardless of the addition of Baf. A1, while the fluorescence of pHLys Green decreased due to lysosomal neutralization caused by the addition of Baf. A1. This indicates that Baf. A1 does not affect lysosomal mass, although lysosomal function is reduced.