Three-dimensional cell culture conditions affect the proteome of cancer-associated fibroblasts

In vitro cell culture systems are an invaluable tool for cell biological research to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns about the reliability and conferrability of the obtained results. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, for example, in pharmacological screens. Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigel amenable to stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAF) on 3D culture conditions. Both, control cells and CAF, change their proteomic composition based on the culture conditions. Critically, cell type differences observed in 2D are often not preserved in 3D, which commonly closer resemble phenotypes observed in vivo. Especially, extracellular matrix and plasma membrane proteins are differentially regulated in 2D versus 3D

A systems study reveals concurrent activation of AMPK and mTOR by amino acids

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKK beta). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes