Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells

Exploring single-cell metabolism and its control on cell growth signals using fluorescent biosensors.

In current cellular biology, we often assume that cells operate at a near steady state. Thisassumption implies that each individual cell performs the same processes at any particular moment. However, this assumption proves to be difficult to reconcile with cellular processes that are dynamic and asynchronous, such as the cell cycle, in which each cell has its own clock, or ey signaling processes such as the MAPK and AKT pathways, which are heterogeneous and dynamic from cell to cell. These heterogeneities play an essential role in cell fate decisions, including proliferation and differentiation. Cell signaling information also ‘spreads’ to other pathways and thus creates complex changes in cellular states, including in metabolic flux and gene expression. The connection between cell signaling and cell metabolism raises the question of whether cell metabolism is heterogeneous too. Furthermore, metabolic states could influence how cells respond to growth signaling cues. In the first part of this dissertation, I explore the question of whether cellular metabolism is heterogeneous in cell populations. I utilize a fluorescence-based FRET biosensor to probe AMPK activity when cellular oxidative phosphorylation (OXPHOS) is inhibited. I show that, in fact, at a single-cell level, cells do not utilize OXPHOS equally, and cellular adaptation after OXPHOS inhibition never reaches a steady state. In the second part, I expand the idea of single-cell metabolism and ask how cell metabolism regulates growth signals at the single-cell level. I developed transposase-based transfection systems that would allow expression of up to three fluorescence biosensors in one transfection to achieve this goal. I also developed an unsupervised clustering technique for multidimensional time-series data to analyze more than 300,000 single cell traces. I showed that the signaling activity under metabolic conditions is heterogeneous even in the same type of metabolic stress. However, the signaling landscape is not infinite since there are only about 30 modes ofviii responses. This study characterizes the complex interaction between cell metabolism and cellular growth signals

Exploring single-cell metabolism and its control on cell growth signals using fluorescent biosensors.

In current cellular biology, we often assume that cells operate at a near steady state. Thisassumption implies that each individual cell performs the same processes at any particular moment. However, this assumption proves to be difficult to reconcile with cellular processes that are dynamic and asynchronous, such as the cell cycle, in which each cell has its own clock, or ey signaling processes such as the MAPK and AKT pathways, which are heterogeneous and dynamic from cell to cell. These heterogeneities play an essential role in cell fate decisions, including proliferation and differentiation. Cell signaling information also ‘spreads’ to other pathways and thus creates complex changes in cellular states, including in metabolic flux and gene expression. The connection between cell signaling and cell metabolism raises the question of whether cell metabolism is heterogeneous too. Furthermore, metabolic states could influence how cells respond to growth signaling cues. In the first part of this dissertation, I explore the question of whether cellular metabolism is heterogeneous in cell populations. I utilize a fluorescence-based FRET biosensor to probe AMPK activity when cellular oxidative phosphorylation (OXPHOS) is inhibited. I show that, in fact, at a single-cell level, cells do not utilize OXPHOS equally, and cellular adaptation after OXPHOS inhibition never reaches a steady state. In the second part, I expand the idea of single-cell metabolism and ask how cell metabolism regulates growth signals at the single-cell level. I developed transposase-based transfection systems that would allow expression of up to three fluorescence biosensors in one transfection to achieve this goal. I also developed an unsupervised clustering technique for multidimensional time-series data to analyze more than 300,000 single cell traces. I showed that the signaling activity under metabolic conditions is heterogeneous even in the same type of metabolic stress. However, the signaling landscape is not infinite since there are only about 30 modes ofviii responses. This study characterizes the complex interaction between cell metabolism and cellular growth signals

Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells

Cell-to-cell heterogeneity in metabolism plays an unknown role in physiology and pharmacology. To functionally characterize cellular variability in metabolism, we treated cells with inhibitors of oxidative phosphorylation (OXPHOS) and monitored their responses with live-cell reporters for ATP, ADP/ATP, or activity of the energy-sensing kinase AMPK. Across multiple OXPHOS inhibitors and cell types, we identified a subpopulation of cells resistant to activation of AMPK and reduction of ADP/ATP ratio. This resistant state persists transiently for at least several hours and can be inherited during cell divisions. OXPHOS inhibition suppresses the mTORC1 and ERK growth signaling pathways in sensitive cells, but not in resistant cells. Resistance is linked to a multi-factorial combination of increased glucose uptake, reduced protein biosynthesis, and G0/G1 cell-cycle status. Our results reveal dynamic fluctuations in cellular energetic balance and provide a basis for measuring and predicting the distribution of cellular responses to OXPHOS inhibition

Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis.

mTORC1 senses nutrients and growth factors and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. These functions position mTORC1 as a central controller of cellular homeostasis, but the behavior of this system in individual cells has not been well characterized. Here, we provide measurements necessary to refine quantitative models for mTORC1 as a metabolic controller. We developed a series of fluorescent protein-TFEB fusions and a multiplexed immunofluorescence approach to investigate how combinations of stimuli jointly regulate mTORC1 signaling at the single-cell level. Live imaging of individual MCF10A cells confirmed that mTORC1-TFEB signaling responds continuously to individual, sequential, or simultaneous treatment with amino acids and the growth factor insulin. Under physiologically relevant concentrations of amino acids, we observe correlated fluctuations in TFEB, AMPK, and AKT signaling that indicate continuous activity adjustments to nutrient availability. Using partial least squares regression modeling, we show that these continuous gradations are connected to protein synthesis rate via a distributed network of mTORC1 effectors, providing quantitative support for the qualitative model of mTORC1 as a homeostatic controller and clarifying its functional behavior within individual cells

Selective Tropism of Dengue Virus for Human Glycoprotein Ib

Abstract Since the hemorrhage in severe dengue seems to be primarily related to the defect of the platelet, the possibility that dengue virus (DENV) is selectively tropic for one of its surface receptors was investigated. Flow cytometric data of DENV-infected megakaryocytic cell line superficially expressing human glycoprotein Ib (CD42b) and glycoprotein IIb/IIIa (CD41 and CD41a) were analyzed by our custom-written software in MATLAB. In two-dimensional analyses, intracellular DENV was detected in CD42b+, CD41+ and CD41a+ cells. In three-dimensional analyses, the DENV was exclusively detected in CD42b+ cells but not in CD42b− cells regardless of the other expressions. In single-cell virus-protein analyses, the amount of DENV was directly correlated with those of CD42b at the Pearson correlation coefficient of 0.9. Moreover, RT- PCR and apoptosis assays showed that DENV was able to replicate itself and release its new progeny from the infected CD42b+ cells and eventually killed those cells. These results provide evidence for the involvement of CD42b in DENV infection

Cellular transformation promotes the incorporation of docosahexaenoic acid into the endolysosome-specific lipid bis(monoacylglycerol)phosphate in breast cancer

Bis(monoacylglycero)phosphates (BMPs), a class of lipids highly enriched within endolysosomal organelles, are key components of the lysosomal intraluminal vesicles responsible for activating sphingolipid catabolic enzymes. While BMPs are understudied relative to other phospholipids, recent reports associate BMP dysregulation with a variety of pathological states including neurodegenerative diseases and lysosomal storage disorders. Since the dramatic lysosomal remodeling characteristic of cellular transformation could impact BMP abundance and function, we employed untargeted lipidomics approaches to identify and quantify BMP species in several in vitro and in vivo models of breast cancer and comparative non-transformed cells and tissues. We observed lower BMP levels within transformed cells relative to normal cells, and consistent enrichment of docosahexaenoic acid (22:6) fatty acyl chain-containing BMP species in both human- and mouse-derived mammary tumorigenesis models. Our functional analysis points to a working model whereby 22:6 BMPs serve as reactive oxygen species scavengers in tumor cells, protecting lysosomes from oxidant-induced lysosomal membrane permeabilization. Our findings suggest that breast tumor cells might divert polyunsaturated fatty acids into BMP lipids as part of an adaptive response to protect their lysosomes from elevated reactive oxygen species levels, and raise the possibility that BMP-mediated lysosomal protection is a tumor-specific vulnerability that may be exploited therapeutically

Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells