PuSH - Publication Server of Helmholtz Zentrum München

Wu, X.* ; Schneider, N.* ; Platen, A.* ; Mitra, I.* ; Blazek, M.* ; Zengerle, R.* ; Schüle, R.* ; Meier, M.

In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip.

Proc. Natl. Acad. Sci. U.S.A. 113, E4143-E4150 (2016)
DOI PMC
Creative Commons Lizenzvertrag
Mammalian target of rapamycin (mTOR) is a central kinase integrating nutrient, energy, and metabolite signals. The kinase forms two distinct complexes: mTORC1 and mTORC2. mTORC1 plays an essential but undefined regulatory function for regeneration of adipose tissue. Analysis of mTOR in general is hampered by the complexity of regulatory mechanisms, including protein interactions and/or phosphorylation, in an ever-changing cellular microenvironment. Here, we developed a microfluidic large-scale integration chip platform for culturing and differentiating human adipose-derived stem cells (hASCs) in 128 separated microchambers under standardized nutrient conditions over 3 wk. The progression of the stem cell differentiation was measured by determining the lipid accumulation rates in hASC cultures. For in situ protein analytics, we developed a multiplex in situ proximity ligation assay (mPLA) that can detect mTOR in its two complexes selectively in single cells and implemented it on the same chip. With this combined technology, it was possible to reveal that the mTORC1 is regulated in its abundance, phosphorylation state, and localization in coordination with lysosomes during adipogenesis. High-content image analysis and parameterization of the in situ PLA signals in over 1 million cells cultured on four individual chips showed that mTORC1 and lysosomes are temporally and spatially coordinated but not in its composition during adipogenesis.
Altmetric
Additional Metrics?
Edit extra informations Login
Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Adipogenesis ; Mtorc1 Regulation ; Microfluidics ; Multiplexed Pla ; Stem Cell Differentiation
ISSN (print) / ISBN 0027-8424
e-ISSN 1091-6490
Quellenangaben Volume: 113, Issue: 29, Pages: E4143-E4150 Article Number: , Supplement: ,
Publisher National Academy of Sciences
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Helmholtz Pioneer Campus (HPC)