Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.

We have recently demonstrated that the rate-limiting enzymes in human glucose metabolism organize into cytoplasmic clusters to form a multienzyme complex, the glucosome, in at least three different sizes. Quantitative high-content imaging data support a hypothesis that the glucosome clusters regulate the direction of glucose flux between energy metabolism and building block biosynthesis in a cluster size-dependent manner. However, direct measurement of their functional contributions to cellular metabolism at subcellular levels has remained challenging. In this work, we develop a mathematical model using a system of ordinary differential equations, in which the association of the rate-limiting enzymes into multienzyme complexes is included as an essential element. We then demonstrate that our mathematical model provides a quantitative principle to simulate glucose flux at both subcellular and population levels in human cancer cells. Lastly, we use the model to simulate 2-deoxyglucose-mediated alteration of glucose flux in a population level based on subcellular high-content imaging data. Collectively, we introduce a new mathematical model for human glucose metabolism, which promotes our understanding of functional roles of differently sized multienzyme complexes in both single-cell and population levels.

Sequential metabolic enzymes have long been hypothesized to form multienzyme metabolic complexes to regulate metabolic flux in cells. Although in vitro biochemistry has not been much fruitful to support the hypothesis, advanced biophysical technologies have successfully resurrected the hypothesis with compelling experimental evidence. As biochemistry has always evolved along with technological advancement over the century (e.g. recombinant protein expression, sitedirected mutagenesis, advanced spectroscopic and structural biology techniques, etc), there has been growing interests in advanced imaging-based biophysical methods to explore enzymes inside living cells. In this work, we describe how we visualize two phase-separated biomolecular condensates of multienzyme metabolic complexes that are associated with de novo purine biosynthesis and glucose metabolism in living human cells and how imaging-based data are quantitatively analyzed to advance our knowledge of enzymes and their assemblies in living cells. Therefore, we envision that the framework we describe here would be the starting point to investigate other metabolic enzymes and their assemblies in various cell types with an unprecedented potential to comprehend enzymes and their network in native habitats.

Glucose metabolism is biochemically intertwined between energy metabolism and building block biosynthesis in living cells. However, it has not been investigated yet how its metabolic network is orchestrated to govern glucose flux in space and time. Since we reported that human enzymes in glucose metabolism are spatially organized into metabolically active membraneless compartments (i.e., glucosomes), we have employed lattice light sheet microscopic imaging and other biophysical and biochemical techniques to understand their functional significance in cellular metabolism. Now, we demonstrated that glucosome assemblies behave like liquid droplets in human cells and thus reversibly respond to environmental changes. In addition, we characterized a molecular architecture of the glucosome, which appears to be constructed from higher-ordered oligomeric structures of its scaffolder enzyme along with transient enzyme-enzyme interactions. Importantly, we found that enzymatic compositions of glucosomes are altered when they are spatially in proximity to mitochondria to functionally couple glycolysis with mitochondrial metabolism in human cells. Collectively, we envision that the subcellular localization-function relationship between glucosomes and mitochondria may represent one of fundamental principles by which 4-dimensional metabolic networks are not only dynamically but also efficiently regulated in living human cells.One Sentence SummaryInvestigation of a 4D functional network of glucose metabolism uncovers a fundamental principal of subcellular metabolic regulation in human cells.

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