The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

Abstract

IntroductionCellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different.ResultsStructurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex.SummaryWe have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance.

Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

“…Recently Duarte et al proposed a role for histone H3.3 clipping in down-regulation of cell cycle-promoting genes during senescence in human fibroblasts (30). Previously we demonstrated that proliferation rate of hepatocarcinoma cells constitutively decreases during the cultivation in 3D culture and after 21 days of growth the majority of cells are either arrested in G 1 or have entered G o phase (31). We now show that the abundance of clipped H2B and H3 consistently increases in C3A cells during their cultivation in 3D culture.…”

A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes

“…With the growing interest in 3D models, the development of various biomaterials has seen exponential growth over the past decade, becoming highly prevalent in oncology (Sittampalam et al, 2015) while other fields are lagging behind, like adipose tissue, despite the fact that proteomics analysis performed by Wrzesinski et al (2014) have shown that expression of proteins involved in fatty acid metabolism can be increased in 3D culture. With the growing interest in 3D models, the development of various biomaterials has seen exponential growth over the past decade, becoming highly prevalent in oncology (Sittampalam et al, 2015) while other fields are lagging behind, like adipose tissue, despite the fact that proteomics analysis performed by Wrzesinski et al (2014) have shown that expression of proteins involved in fatty acid metabolism can be increased in 3D culture.…”

“…Due to their tunable mechanical and chemical properties, hydrogels possess biophysical characteristics similar to natural tissue and represent highly effective matrices for 3D cell culture. With the growing interest in 3D models, the development of various biomaterials has seen exponential growth over the past decade, becoming highly prevalent in oncology (Sittampalam et al, 2015) while other fields are lagging behind, like adipose tissue, despite the fact that proteomics analysis performed by Wrzesinski et al (2014) have shown that expression of proteins involved in fatty acid metabolism can be increased in 3D culture. In addition to include adipose ECM components, the mechanical properties of our HAbased scaffold display a Young's modulus of 0.5 kPa, which is in the same range of adipose tissue ECM, whose biomechanical measurements have been performed without any decellularization process, giving values of Young's modulus between 1.5 and 3 kPa (Alkhouli et al, 2013).…”

Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

“…no. 15400-054), diluted 1:4 and sown out into falcon tubes or microtitre plates [56]. Cells were collected 5 days after trypsinization at 85% Zr-IMAC for phosphopeptide enrichment in phosphoproteomics confluence at subculture 10 and washed 5 times with warm (37 o C) Hanks' Balanced Salt Solution (HBSS without Ca++ and Mg++, Gibco Cat.…”