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

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 adiposederived 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.microfluidics | stem cell differentiation | adipogenesis | mTORC1 regulation | multiplexed PLA

MicroRNA-496 and Mechanistic Target of Rapamycin Expression are Associated with Type 2 Diabetes Mellitus and Obesity in Elderly People

“…Accordingly, we observed significant downregulation of miR-496 with increasing obesity stages, suggesting a role of miR-496 also in the BMI-dependent regulation of mTOR. These findings are in line with our understanding that T2DM and obesity are strongly linked to the mTORC1 signaling pathway and dysfunctional adipose tissue [17][18][19]. Hence, mTOR is reported to regulate adipogenesis, which is vital for animals to maintain energy balance by storing lipids.…”

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

“…IEF separation occurs when an electric field is applied along the axis of a stable pH gradient, commonly formed by polyprotic amphoteric buffers called carrier ampholytes (CAs) . Post‐translational modifications such as ubiquitination, phosphorylation, and truncation are of functional biological importance and also serve as biomarkers for disease . Because post‐translational modifications typically result in small molecular mass changes , the modifications are challenging to resolve using size‐based separations.…”

Multiplexed Point-of-Care Testing – xPOCT

“…This technology combines rapid and facile manufacturing with high-throughput multiplexing, using a minimum number of channel inputs. Its applicability for simultaneous multianalyte measurements in proteomics 64 , 65 , cellomics [66] and genomics [67] was demonstrated. Although they have a great potential for high-level multiplexing, mLSI-based systems are yet not applicable to xPOCT due to their system complexity, as well as bulky instrumentation for the pneumatic control and the respective fluid connection.…”