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Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):E4143-50. doi: 10.1073/pnas.1601207113. Epub 2016 Jul 5.

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

Author information

1
Microfluidic and Biological Engineering, Department of Microsystems Engineering (IMTEK), University of Freiburg, 79110 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany;
2
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany; Laboratory for MEMS Applications, Department of Microsystems Engineering (IMTEK), University of Freiburg, 79110 Freiburg, Germany;
3
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany; Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, 79106 Freiburg, Germany; Deutsches Konsortium für Translational Krebsforschung, Standort Freiburg, 79106 Freiburg, Germany.
4
Microfluidic and Biological Engineering, Department of Microsystems Engineering (IMTEK), University of Freiburg, 79110 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany; matthias.meier@imtek.de.

Abstract

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.

KEYWORDS:

adipogenesis; mTORC1 regulation; microfluidics; multiplexed PLA; stem cell differentiation

PMID:
27382182
PMCID:
PMC4961165
DOI:
10.1073/pnas.1601207113
[Indexed for MEDLINE]
Free PMC Article

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