Biology subjects

Schmitt, D. L.

Publications and source records attributed to Schmitt, D. L..

12 recordsLinked to original sources

bioRxiv2026

Revealing the spatiotemporal dynamics of methionine metabolism with a genetically encoded single-fluorophore biosensor

Methionine is an essential amino acid, used for protein synthesis, redox homeostasis, and methylation reactions throughout the cell. However, the compartmentalized dynamics of methionine have remained elusive, due to a lack of available tools to measure methionine with high spatial and temporal resolution. To address this limitation, we have developed a single fluorescent protein-based methionine optical reporter (Meteor) which reports subcellular changes in methionine with high dynamic range. Using Meteor, we demonstrate the subcellular uptake of methionine in multiple cell lines into several locations, including the mitochondrial matrix. Furthermore, we use Meteor to illuminate the dynamics of the methionine cycle in the cytoplasm and nucleus, finding cancer cells can rapidly increase methionine from metabolic precursors in both locations. Finally, demonstrated that Meteor can be used to visualize methionine dynamics in vivo using Caenorhabditis elegans. Thus, we have developed a new tool to measure methionine dynamics across scales with high dynamic range and spatiotemporal resolution.

biochemistry↗

bioRxiv2026

Illuminating spatial dynamics of glutamine metabolism with a sensitive genetically encoded biosensor

Glutamine is the most abundant amino acid in serum, used as a key nutrient by cells for protein synthesis, energy production, carbon and nitrogen metabolism, and cellular redox balance. The use of glutamine in the cell is highly compartmentalized, but the dynamics of glutamine metabolism across organelles and individual cells are not fully understood. To illuminate subcellular glutamine dynamics, we developed a green fluorescent protein-based intracellular glutamine optical reporter, iGlo. We find iGlo is sensitive and specific for glutamine and can be used to measure glutamine uptake, production, and consumption with high spatiotemporal resolution in multiple cell types. Furthermore, multiplexed imaging of iGlo with a lactate biosensor in single cells reveals the temporal crosstalk between glucose and glutamine metabolism to maintain energy homeostasis. Thus, iGlo enables the sensitive and precise study of compartmentalized glutamine dynamics and represents a new and enhanced tool for studying the spatiotemporal dynamics and regulation of metabolism.

biochemistry↗

bioRxiv2025

Spatial regulation of AMPK activity under oxidative stress requires LKB1

AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, with over 100 identified downstream targets throughout the cell. In response to cellular stress, including energetic stress, AMPK is activated via binding of AMP and phosphorylation by upstream kinases, including liver kinase B1 (LKB1). We and others have found that the activation of AMPK in response to cellular stress has distinct subcellular mechanisms, indicating compartmentalized regulation of AMPK signaling. Oxidative stress is known to stimulate AMPK activity, but how AMPK is spatially regulated by oxidative stress is underexplored. Using a single-fluorophore excitation-ratiometric AMPK activity reporter (ExRai AMPKAR), we find that oxidative stress induced by hydrogen peroxide (H2O2) results in AMPK activity with distinct spatiotemporal dynamics. We found that in the cytoplasm, nucleus, outer mitochondrial membrane, and cytosolic lysosomal surface, phosphorylation of AMPK by LKB1 is required for AMPK activity. Using a biosensor for ATP, we found at the cytoplasm and lysosome local ATP depletion dictates kinetics of AMPK activity. Using a multi- omics approach, we discover that in response to oxidative stress, AMPK mediates significant metabolic and gene expression changes, including upregulation of oxidative stress response through nuclear factor erythroid 2-related factor 2 (NRF2). Expanding on this identified mechanism, we find that non-small cell lung cancers harboring Kelch-like ECH-associated protein 1 (KEAP1) mutations have a functionally deficient LKB1-AMPK signaling network in response to oxidative stress. Altogether, this work provides new insights into how the subcellular environment influences localized AMPK activity, and identifies how AMPK regulates the cellular response to oxidative stress.

biochemistry↗

bioRxiv2025

Visualizing PINK1 Activity Dynamics in Single Cells with a Phase Separation-Based Kinase Activity Reporter

Phosphatase and tensin homologue-induced kinase 1 (PINK1) is a serine/threonine kinase that plays roles in mitophagy, cell death, and regulation of cellular bioenergetics. Current approaches for studying PINK1 function depend on bulk techniques that can only provide snapshots of activity and could miss the dynamics and cell-to-cell heterogeneity of PINK1 activity. Therefore, we sought to develop a novel PINK1 kinase activity reporter to characterize PINK1 activity. Taking advantage of the separation of phases-based activity reporter of kinase (SPARK) design, we developed a phase separation-based PINK1 biosensor (PINK1-SPARK). With PINK1-SPARK, we observe real-time PINK1 activity in single cells treated with mitochondria depolarizing agents or pharmacological activators. We then developed a Halo Tag-based PINK1-SPARK for multiplexed imaging of PINK1 activity with live-cell markers of mitochondrial damage. Thus, PINK1-SPARK is a new tool that enables temporal measurement of PINK1 activity in single live cells, allowing for further elucidation of the role of PINK1 in mitophagy and cell function.

cell biology↗

bioRxiv2025

Neutrophil extracellular traps offer a new therapeutic target for elephant endotheliotropic herpes hemorrhagic disease (EEHV-HD)

Elephant survival is threatened by a devastating hemorrhagic disease called elephant endotheliotropic herpes virus-hemorrhagic disease (EEHV-HD). Once clinical signs are observed in elephants, the disease progresses rapidly and frequently results in death. EEHV-HD negatively impacts elephant conservation because very young, reproductively immature elephants are most at risk for death. Ongoing efforts to understand disease pathogenesis and progression may identify treatment targets and improve clinical outcomes. In some lethal EEHV-HD cases, microthrombosis has been observed in organ tissues similar to other hemorrhagic diseases in humans and animals where sticky webs of protein-coated DNA strands called neutrophil extracellular traps (NETs) exacerbate thrombosis and hemorrhage associated with disseminated intravascular coagulation (DIC). In this study, we sought to identify if NET formation occurs in elephants and could contribute to poor outcomes in EEHV-HD. Our study demonstrated NET release for the first time from elephant heterophils (neutrophils) that occurred in response to various stimuli, including plasma from EEHV-HD affected elephants. EEHV-HD affected tissues contained extensive NETs suggesting that dysregulated NET formation contributes to pathogenesis of this disease. Importantly, elephant neutrophils were blocked from releasing NETs in response to EEHV-HD plasma using known NET inhibitors. The ability to stop NETs in EEHV-HD offers a new therapeutic approach that could be combined with current therapies to improve survival for affected elephants and to positively impact conservation efforts.

immunology↗

bioRxiv2024

A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA

Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on dynamic changes in malonyl-CoA and fatty acid biosynthesis that could be occurring within a single cell. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu (malonyl-CoA intracellular biosensor to understand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in E. coli, observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to sensitively study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis.

biochemistry↗

bioRxiv2023

An expanded palette of ATP sensors for subcellular and multiplexed imaging

Genetically encoded fluorescent biosensors that detect changes in ATP levels in live cells have enabled the discovery of novel roles for ATP in cellular processes and signaling. Many of these available ATP biosensors have a limited dynamic range, or have ATP affinities that are not suitable for sensing the physiological concentrations of ATP in mammalian cells. To address these limitations, we developed a FRET-based ATP biosensor with enhanced dynamic range and signal to noise ratio, eATeam. Using eATeam, we uncovered distinct spatiotemporal dynamics of ATP changes upon inhibition of cellular energy production. We also developed dimerization-dependent GFP and RFP-based ATP biosensors with enhanced dynamic ranges compared to the current standard in the field. Using the single-color ATP biosensors, we visualized the complex interplay between AMPK activity, ATP, lactate, and calcium by multiplexed imaging in single cells. This palette of ATP sensors expands the toolbox for interrogating subcellular ATP regulation and metabolic signaling in living cells.

cell biology↗

bioRxiv2022

High-content quantitative high-throughput screening identifies a cell cycle-associated signaling cascade that regulates a multienzyme metabolic assembly for glucose metabolism

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, the glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism in human cells. We developed a high-content quantitative high-throughput screening (qHTS) assay to evaluate the impact of small molecule libraries on the formation of PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three active compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling cascade that regulates the formation of PFK1-mediated glucosome assembly in human cells.

biochemistry↗

bioRxiv2022

Functional oscillation of a multienzyme glucosome assembly during cell cycle progression

Glucose metabolism has been studied extensively to understand functional interplays between metabolism and a cell cycle. However, our understanding of cell cycle-dependent metabolic adaptation particularly in human cells remains largely elusive. Meanwhile, human enzymes in glucose metabolism are shown to functionally organize into three different sizes of a multienzyme metabolic assembly, the glucosome, to regulate glucose flux in a size-dependent manner. Here, using fluorescence single-cell imaging techniques, we discover that glucosomes spatiotemporally oscillate during a cell cycle in an assembly size-dependent manner. Importantly, their oscillation at single-cell levels is in accordance with functional contributions of glucose metabolism to cell cycle progression at a population level. Collectively, we demonstrate functional oscillation of glucosomes during cell cycle progression and thus their biological significance to human cell biology.

cell biology↗

bioRxiv2021

Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

molecular biology↗

bioRxiv2020

Elephant genomes reveal insights into differences in disease defense mechanisms between species

Disease susceptibility and resistance comprise important factors in conservation, particularly in elephants. To determine genetic mechanisms underlying disease resistance and other unique elephant traits, we estimated 862 and 1,017 potential regulatory elements in Asian and African elephants, respectively. These elements are significantly enriched in both species with differentially expressed genes involved in immunity pathways, including tumor-necrosis factor which plays a role in the response to elephant endotheliotropic herpesvirus (EEHV). Population genomics analyses indicate that amplified TP53 retrogenes are maintained by purifying selection and may contribute to cancer resistance in elephants, including less malignancies in African vs. Asian elephants. Positive selection scans across elephant genomes revealed genes that may control iconic elephant traits such as tusk development, memory, and somatic maintenance. Our study supports the hypothesis that interspecies variation in gene regulation contributes to differential inflammatory responses leading to increased infectious disease and cancer susceptibility in Asian versus African elephants. Genomics can inform functional immunological studies which may improve both conservation for elephants and human therapies.

evolutionary biology↗

bioRxiv2019

Computational modeling reveals frequency modulation of calcium-cAMP/PKA pathway in dendritic spines

Dendritic spines are the primary excitatory postsynaptic sites that act as subcompartments of signaling. Ca2+ is often the first and most rapid signal in spines. Downstream of calcium, the cAMP/PKA pathway plays a critical role in the regulation of spine formation, morphological modifications, and ultimately, learning and memory. While the dynamics of calcium are reasonably well-studied, calcium-induced cAMP/PKA dynamics, particularly with respect to frequency modulation, are not fully explored. In this study, we present a well-mixed model for the dynamics of calcium-induced cAMP/PKA dynamics in dendritic spines. The model is constrained using experimental observations in the literature. Further, we measured the calcium oscillation frequency in dendritic spines of cultured hippocampal CA1 neurons and used these dynamics as model inputs. Our model predicts that the various steps in this pathway act as frequency modulators for calcium and the high frequency of calcium input is filtered by AC1 and PDEs in this pathway such that cAMP/PKA only responds to lower frequencies. This prediction has important implications for noise filtering and long-timescale signal transduction in dendritic spines. A companion manuscript presents a three-dimensional spatial model for the same pathway.\n\nStatement of SignificancecAMP/PKA activity triggered by calcium is an essential biochemical pathway for synaptic plasticity, regulating spine structure, and long-term potentiation. In the current study, we predicted that for a given calcium input, AC1, and PDE1 kinetics reflect both the high and the low frequencies with different amplitudes and cAMP/PKA acts as a leaky integrator of calcium because of frequency attenuation by the intermediary steps. These findings have implications for cAMP/PKA signaling in dendritic spines in particular and neuronal signal transduction in general.

biophysics↗