SPATIAL AND FUNCTIONAL ANALYSIS OF MEMBRANELESS ORGANELLES IN GLUCOSE METABOLIC NETWORK USING LIGHT SHEET MICROSCOPY
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Author/Creator ORCID
Date
2021-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Chemistry
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Distribution Rights granted to UMBC by the author.
Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan thorugh a local library, pending author/copyright holder's permission.
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Abstract
Metabolic enzymes can arrange into multi-enzyme complexes which regulate metabolic processes. In the glucose metabolism, this multi-enzyme complex has been termed the ‘glucosome'. Recently, glucosomes have been reported to direct glucose flux into various other pathways in live human cells, with small-sized glucosomes directing the metabolism through the entirety of glycolysis. However, projected images generated by conventional microscopies are limited in detecting the small-sized glucosomes due to the contrast of the glucosomes against a projected background. Therefore, we have utilized 3-dimensional lattice-light sheet microscopy to observe glucosomes in live cells with better spatial resolution. This analysis led to the characterization of relative density inside of glucosomes which determined that the characteristic concentration of one enzyme, phosphofructokinase-liver type, was dependent on the size of the glucosome, but another enzyme, pyruvate kinase (isoform 2) was independent of size. To better understand the formation of glucosomes, exposing the cells to increased osmotic pressure resulted in an increase in glucosomes via liquid phase separation. With increased osmotic pressure, the quantity and size of glucosomes increased, leading us to conclude that glucosomes are formed through liquid-liquid phase separation and may have an internal structure provided by the oligomeric PFKL. The 3D lattice light sheet microscope also allowed us to observe that a significant portion of glucosomes were in subcellular proximity to the surface of mitochondria. Furthermore, disruptions to the mitochondrial metabolism dramatically affected the small glucosomes that were in proximity with the surface of the mitochondria, demonstrating the functional and spatial relationship shared between glucosomes and mitochondria metabolic compartments. While larger glucosomes were statistically retained, the quantity of small glucosomes associated with the surface of mitochondria were reduced more than 50% when cells were treated with a mitochondria oxidative phosphorylation inhibitor, demonstrating that the physical characteristics of glucosomes are related to their metabolic function inside of cells. Next, glucosomes that were not in proximity to the mitochondria were evaluated for their spatial coordination with another subcellular compartment. The glucosomes were analyzed to observe a high spatial proximity with both microtubules and actin in the cytoskeleton. Larger and small glucosomes were readily associated with the cytoskeleton, which suggests that glucosomes are regulated in a size-dependent matter with other metabolic pathways. Collectively, this work demonstrates a method to spatially analyze the subcellular proximity of metabolic compartments and provides consistent findings that the glucosome metabolic compartment directs glucose flux in a size-dependent manner.