Multi-dimensional Fluorescence Live-Cell Imaging for Glucosome Dynamics in Living Human Cells.

Fluorescence live-cell imaging that has contributed to our understanding of cell biology is now at the frontline of studying quantitative biochemistry in a cell. Particularly, technological advancements of fluorescence live-cell imaging and associated strategies in recent years have allowed us to discover various subcellular macromolecular assemblies in living human cells. Here we describe how real-time dynamics of a multienzyme metabolic assembly, the "glucosome," that is responsible for regulating glucose flux at subcellular levels, has been investigated in both 2- and 3-dimensional space of single human cells. We envision that such multi-dimensional fluorescence live-cell imaging will continue to revolutionize our understanding of how intracellular metabolic pathways and their network are functionally orchestrated at single-cell levels.

Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90.

Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid-liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low-complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Finally, we demonstrate that the heat shock protein 90 KDa helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of heat shock protein 90 KDa activity induced the transformation of formylglycinamidine ribonucleotide synthase clusters into more irregularly shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells.

Phase-separated condensates of metabolic complexes in living cells: Purinosome and glucosome.

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 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, site-directed mutagenesis, advanced spectroscopy and structural biology techniques, etc.), there has been growing interest 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.

Identification of a multienzyme complex for glucose metabolism in living cells.

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.

Effects of systemic inflammation on insulin sensitivity in horses and inflammatory cytokine expression in adipose tissue.

OBJECTIVE: To determine whether an inflammatory challenge induces insulin resistance in horses and examine possible contributions of adipose tissue to inflammatory cytokine production. ANIMALS: 15 adult mares. PROCEDURES: Lipopolysaccharide (0.045 mug/kg, IV) or saline solution was administered, and insulin sensitivity was determined by means of the hyperinsulinemic, euglycemic clamp procedure or an adipose tissue biopsy was performed. Adipose tissue samples were collected, and mature adipocytes were obtained. Mature adipocytes were stimulated with lipopolysaccharide or dedifferentiated into preadipocytes and then stimulated with lipopolysaccharide. Interleukin-1, interleukin-6, and tumor necrosis factor A expression in blood, adipose tissue, and adipocytes was quantified with a real-time, reverse transcriptase- PCR assay. RESULTS: Lipopolysaccharide induced a transient increase in insulin sensitivity followed by a reduction in insulin sensitivity at 24 hours. Increased cytokine expression was observed in blood and adipose tissue following administration of lipopolysaccharide, and adipocytes and preadipocytes stimulated with lipopolysaccharide stained positive for tumor necrosis factor A. Expression of interleukin-1, interleukin-6, and tumor necrosis factor A was detected in preadipocytes stimulated with lipopolysaccharide, and interleukin-6 and tumor necrosis factor A were detected in mature adipocytes stimulated with lipopolysaccharide. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that insulin resistance develops following systemic inflammation in horses and suggested that adipose tissue may contribute to this inflammatory response. Methods to regulate insulin sensitivity may improve clinical outcome in critically ill patients.

Rapid phase adjustment of melatonin and core body temperature rhythms following a 6-h advance of the light/dark cycle in the horse.

BACKGROUND: Rapid displacement across multiple time zones results in a conflict between the new cycle of light and dark and the previously entrained program of the internal circadian clock, a phenomenon known as jet lag. In humans, jet lag is often characterized by malaise, appetite loss, fatigue, disturbed sleep and performance deficit, the consequences of which are of particular concern to athletes hoping to perform optimally at an international destination. As a species renowned for its capacity for athletic performance, the consequences of jet lag are also relevant for the horse. However, the duration and severity of jet lag related circadian disruption is presently unknown in this species. We investigated the rates of re-entrainment of serum melatonin and core body temperature (BT) rhythms following an abrupt 6-h phase advance of the LD cycle in the horse. METHODS: Six healthy, 2 yr old mares entrained to a 12 h light/12 h dark (LD 12:12) natural photoperiod were housed in a light-proofed barn under a lighting schedule that mimicked the external LD cycle. Following baseline sampling on Day 0, an advance shift of the LD cycle was accomplished by ending the subsequent dark period 6 h early. Blood sampling for serum melatonin analysis and BT readings were taken at 3-h intervals for 24 h on alternate days for 11 days. Disturbances to the subsequent melatonin and BT 24-h rhythms were assessed using repeated measures ANOVA and analysis of Cosine curve fitting parameters. RESULTS: We demonstrate that the equine melatonin rhythm re-entrains rapidly to a 6-h phase advance of an LD12:12 photocycle. The phase shift in melatonin was fully complete on the first day of the new schedule and rhythm phase and waveform were stable thereafter. In comparison, the advance in the BT rhythm was achieved by the third day, however BT rhythm waveform, especially its mesor, was altered for many days following the LD shift. CONCLUSION: Aside from the temperature rhythm disruption, rapid resynchronization of the melatonin rhythm suggests that the central circadian pacemaker of the horse may possess a particularly robust entrainment response. The consequences for athletic performance remain unknown.

The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.