PMID- 27382182
OWN - NLM
STAT- MEDLINE
DCOM- 20171127
LR  - 20181113
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Print)
IS  - 0027-8424 (Linking)
VI  - 113
IP  - 29
DP  - 2016 Jul 19
TI  - In situ characterization of the mTORC1 during adipogenesis of human adult stem 
      cells on chip.
PG  - E4143-50
LID - 10.1073/pnas.1601207113 [doi]
AB  - 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.
FAU - Wu, Xuanye
AU  - Wu X
AD  - 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;
FAU - Schneider, Nils
AU  - Schneider N
AD  - 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;
FAU - Platen, Alina
AU  - Platen A
AD  - 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;
FAU - Mitra, Indranil
AU  - Mitra I
AD  - 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;
FAU - Blazek, Matthias
AU  - Blazek M
AD  - 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;
FAU - Zengerle, Roland
AU  - Zengerle R
AD  - 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;
FAU - Schule, Roland
AU  - Schule R
AD  - Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 
      Freiburg, Germany; Urologische Klinik und Zentrale Klinische Forschung, Klinikum 
      der Universitat Freiburg, 79106 Freiburg, Germany; Deutsches Konsortium fur 
      Translational Krebsforschung, Standort Freiburg, 79106 Freiburg, Germany.
FAU - Meier, Matthias
AU  - Meier M
AD  - 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.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20160705
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - EC 2.7.11.1 (Mechanistic Target of Rapamycin Complex 1)
SB  - IM
MH  - Adipogenesis/*physiology
MH  - Adult Stem Cells/*metabolism
MH  - Cells, Cultured
MH  - Humans
MH  - Lab-On-A-Chip Devices
MH  - Lysosomes/metabolism
MH  - Mechanistic Target of Rapamycin Complex 1/*metabolism
PMC - PMC4961165
OTO - NOTNLM
OT  - adipogenesis
OT  - mTORC1 regulation
OT  - microfluidics
OT  - multiplexed PLA
OT  - stem cell differentiation
COIS- The authors declare no conflict of interest.
EDAT- 2016/07/07 06:00
MHDA- 2017/11/29 06:00
PMCR- 2017/01/19
CRDT- 2016/07/07 06:00
PHST- 2016/07/07 06:00 [entrez]
PHST- 2016/07/07 06:00 [pubmed]
PHST- 2017/11/29 06:00 [medline]
PHST- 2017/01/19 00:00 [pmc-release]
AID - 1601207113 [pii]
AID - 201601207 [pii]
AID - 10.1073/pnas.1601207113 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):E4143-50. doi: 
      10.1073/pnas.1601207113. Epub 2016 Jul 5.