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Updated: Mar 1, 2026
A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
Activity-independent targeting of mTOR to lysosomes in primary osteoclasts
Andrew Wang1, Luciene R Carraro-Lacroix1, Celeste Owen2
1Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
Abstract:
Mammalian target of rapamycin (mTOR) is activated by numerous stimuli, including amino acids and growth factors. This kinase is part of the mTOR complex 1 (mTORC1) which regulates cell proliferation, differentiation, and autophagy. Active mTORC1 is located on lysosomes and has been reported to disassociate from the lysosomal surface in the absence of amino acids. Furthermore, mTORC1 activity has been linked to the vacuolar H+-ATPases (V-ATPases), the proton pumps responsible for lysosomal acidification; however, the exact role of the V-ATPases in mTORC1 signaling is not known. To elucidate the mechanisms involved in mTORC1 regulation by the V-ATPases, we used primary osteoclasts derived from mice carrying a point (R740S) mutation in the a3 subunit of the V-ATPase. In these cells, the mutant protein is expressed but the pump is not functional, resulting in higher lysosomal pH. By analyzing mTOR activation, mTOR/lysosome co-localization, and lysosomal positioning using confocal microscopy, fractionation, and ultrapure lysosomal purification methods, we demonstrate that in primary osteoclasts, mTOR is localized on the lysosomal surface even when mTOR activity is inhibited. Our findings reveal that mTOR targeting to the lysosome in osteoclasts is activity-independent, and that its disassociation from the lysosome during starvation is not universal.
Insights
Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) targets lysosomes independently of its activity. This challenges the idea that mTORC1 universally disassociates from lysosomes during starvation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a key regulator of cell growth, proliferation, and autophagy.
- mTORC1 activity is linked to lysosomal acidification mediated by vacuolar H+-ATPases (V-ATPases), but the precise role of V-ATPases in mTORC1 signaling remains unclear.
- mTORC1 is typically found on lysosomes and is thought to disassociate during amino acid starvation.
Purpose of the Study:
- To investigate the role of V-ATPases in regulating mTORC1 localization and activity.
- To determine if mTORC1 disassociation from lysosomes during starvation is a universal mechanism.
Main Methods:
- Utilized primary osteoclasts from mice with a V-ATPase a3 subunit mutation (R740S) leading to impaired lysosomal acidification.
- Employed confocal microscopy, cell fractionation, and lysosome purification to analyze mTOR localization and activity.
- Assessed mTOR/lysosome co-localization and lysosomal positioning.
Main Results:
- In osteoclasts with defective V-ATPases, mTOR remained localized on the lysosomal surface even when mTOR activity was inhibited.
- Demonstrated that mTOR targeting to lysosomes in osteoclasts is independent of mTOR kinase activity.
- Found that mTORC1 does not universally disassociate from lysosomes during starvation in these cells.
Conclusions:
- Lysosomal V-ATPases do not appear to regulate mTORC1 targeting to the lysosome in osteoclasts.
- mTORC1 localization to lysosomes in osteoclasts is an activity-independent process.
- The disassociation of mTORC1 from lysosomes during starvation is not a universal phenomenon.
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