Inferring mitochondrial and cytosolic metabolism by coupling isotope tracing and deconvolution.

The inability to inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Previous work addressed this challenge by analyzing metabolism in isolated organelles, which grossly bias metabolic activity. Here, we describe a method for inferring physiological metabolic fluxes and metabolite concentrations in mitochondria and cytosol based on isotope tracing experiments performed with intact cells. This is made possible by computational deconvolution of metabolite isotopic labeling patterns and concentrations into cytosolic and mitochondrial counterparts, coupled with metabolic and thermodynamic modelling. Our approach lowers the uncertainty regarding compartmentalized fluxes and concentrations by one and three orders of magnitude compared to existing modelling approaches, respectively. We derive a quantitative view of mitochondrial and cytosolic metabolic activities in central carbon metabolism across cultured cell lines without performing cell fractionation, finding major variability in compartmentalized malate-aspartate shuttle fluxes. We expect our approach for inferring metabolism at a subcellular resolution to be instrumental for a variety of studies of metabolic dysfunction in human disease and for bioengineering.

Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability.

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

Ice Scallops: A Laboratory Investigation of the Ice-Water Interface.

Ice scallops are a small-scale (5-20cm) quasi-periodic ripple pattern that occurs at the ice-water interface. Previous work has suggested that scallops form due to a self-reinforcing interaction between an evolving ice-surface geometry, an adjacent turbulent flow field, and the resulting differential melt rates that occur along the interface. In this study, we perform a series of laboratory experiments in a refrigerated flume to quantitatively investigate the mechanisms of scallop formation and evolution in high resolution. Using particle-image velocimetry, we probe an evolving ice-water boundary layer at sub-millimeter scales and 15Hz frequency. Our data reveals three distinct regimes of ice-water interface evolution: A transition from flat to scalloped ice; an equilibrium scallop geometry; and an adjusting scallop interface. We find that scalloped ice geometry produces a clear modification to the ice-water boundary layer, characterized by a time-mean recirculating eddy feature that forms in the scallop trough. Our primary finding is that scallops form due to a self reinforcing feedback between the ice-interface geometry and shear production of turbulent kinetic energy in the flow interior. The length of this shear production zone is therefore hypothesized to set the scallop wavelength.