Biology subjects

Ko, B.

Publications and source records attributed to Ko, B..

13 recordsLinked to original sources

bioRxiv2026

Simultaneous brain-wide single-cell recording resolves spatiotemporal memory architecture

Many fundamental mammalian brain functions emerge from the coordinated activity of cells distributed across large, brain-wide networks. To understand these processes in healthy and diseased states, ideally one would simultaneously measure and analyze single-cell activity at the brain-wide scale, an enduring challenge for live-measurement approaches that often face an inherent tradeoff between spatial resolution and scale. Here, we present GLOBE (sinGle-cell spatiotemporaL recOrding Brain-widE), a technology for brain-wide single-cell recording of cellular activity in vivo with spatiotemporal resolution, physiological sensitivity, and parallelization-accelerated readout. GLOBE leverages genetically encoded intracellular protein tape recorders and a high-throughput computational platform for integrated image and signal analysis. GLOBE records analog signal amplitudes across a continuous time axis, requires only standard light microscopy for in situ readout, and is compatible with expansion microscopy and RNA readouts. We applied GLOBE to simultaneously record transcriptional activity of the immediate early gene Fos in up to 219,703 neurons simultaneously across a single mouse brain over 5.5 continuous days, with a timestamp precision of 3.1-6.7 hours (median absolute error), a local recording density of 69-90% of neurons per imaging field of view, and a post-mortem imaging readout speed of 2.9 seconds per neuron on average. GLOBE resolves the brain-wide spatiotemporal structure of single-cell activity, revealing that Fos transcriptional dynamics associated with fear learning and memory retrieval are distributed across the brain with region-specific temporal heterogeneity, and that the variance of this structure scales down as the number of sampled cells increases. We envision GLOBE to have broad applications for dissecting and decoding physiological and pathological processes at the brain-wide scale.

neuroscience↗

bioRxiv2025

Multiplexed, scalable analog recording of gene regulation dynamics over weeks using intracellular protein tapes

Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape--a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed in situ recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-fos, Arc, Egr1, and Npas4 activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.

synthetic biology↗

bioRxiv2025

IN4MER Bioink: A Phosphorescent Biosensing Bio-ink for Multiple Analytes (Glucose, Lactate, Oxygen) Measurements and Temperature Sensing Applications

3D bioprinting has revolutionized tissue engineering by enabling researchers to create much more complex structures than was practical with earlier techniques. Bioprinting uses computer-controlled layer-by-layer deposition of a mixture of hydrogels and living cells and the resulting structures can mimic the complex geometries of many living tissues by incorporating multiple bioinks with varied material properties and cell populations, allowing researchers to design structures that vary not only in shape, but also in mechanical, chemical, and biological properties throughout the bioprinted construct. However, techniques for evaluating these living constructs and monitoring them over time have not yet caught up to these innovations. Here we describe a novel approach to reporting nutrient values in real-time throughout the scaffold itself, accomplished by dispersing oxygen, glucose, and lactate sensitive microspheres within bioinks. These can be noninvasively interrogated using low-cost phosphorescence lifetime readers to determine and track nutrient concentrations across our bioprinted constructs in real time. The wealth of information this technique produces suggests this may provide a powerful new tool for evaluating and designing future bioprinted constructs.

bioengineering↗

bioRxiv2024

Chemotagging: a chemogenetic approach for identifying cell types with in vivo calcium imaging

The ability to monitor the activity of specific cell types in vivo is critical for understanding the complex interplay between various neuronal populations driving freely moving behavior. Existing methods, such as optogenetic tagging (i.e., Optotagging1), have proven useful for identifying cell types in in vivo electrophysiological recordings during freely moving behavior. However, electrophysiological recordings are limited in their capacity to track the same neuronal populations across long periods of time (days to weeks). Single-photon miniscope imaging offers the advantage of tracking the same cells across weeks to months; however, it is difficult to distinguish different cell types within the recorded population. Here, we present "chemotagging," a technique that allows for the identification of specific cell types in in vivo calcium imaging recordings. This protocol offers a method for tagging cell types with chemogenetic tools like Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)2, while simultaneously recording calcium activity from a pan-neuronal population with calcium indicators. We highlight the key advantages and limitations of chemotagging and its potential implications for neuroscience research.

neuroscience↗

bioRxiv2024

An anterior hypothalamic circuit gates stress vulnerability

Prior adversity increases susceptibility to subsequent stressful events, but the causal underlying changes in brain circuitry are poorly understood. We harnessed unbiased whole-brain activity mapping to identify circuits that are functionally remodeled by prior adversity. This revealed that the anterior hypothalamic nucleus (AHN) displays heightened stress reactivity in previously stressed mice. This was accompanied by increased functional connectivity between the AHN and a threat-related limbic network. Using in vivo Miniscope imaging, we found that neuronal activity in the AHN encodes stressor valence. Moreover, stimulating AHN neurons enhanced, and inhibiting their activity mitigated, reactivity to stressful events. Lastly, silencing amygdala inputs to the AHN abolished the ability of prior adversity to increase stress sensitivity. These findings define a key role of the AHN in gating stress vulnerability by scaling valence signals from the amygdala.

neuroscience↗

bioRxiv2024

Mild activation of the mitochondrial unfolded protein response increases lifespan without increasing resistance to stress

The mitochondrial unfolded protein response (mitoUPR) is a stress response pathway that responds to mitochondrial insults by altering gene expression to recover mitochondrial homeostasis. The mitoUPR is mediated by the stress-activated transcription factor ATFS-1. Constitutive activation of ATFS-1 increases resistance to exogenous stressors but paradoxically decreases lifespan. In this work, we determined the optimal levels of expression of activated ATFS-1 with respect to lifespan and resistance to stress by treating constitutively-active atfs-1(et17) worms with different concentrations of RNA interference (RNAi) bacteria targeting atfs-1. We observed the maximum lifespan of atfs-1(et17) worms at full-strength atfs-1 RNAi, which was significantly longer than wild-type lifespan. Under the conditions of maximum lifespan, atfs-1(et17) worms did not show enhanced resistance to stress, suggesting a trade-off between stress resistance and longevity. The maximum resistance to stress in atfs-1(et17) worms occurred on empty vector (0% atfs-1 knockdown). Under these conditions, atfs-1(et17) worms are short-lived. This indicates that constitutive activation of ATFS-1 can increase lifespan or enhance resistance to stress but not both, at the same time. Finally, we determined the timing requirements for ATFS-1 to affect lifespan. We found that knocking down atfs-1 expression only during development is sufficient to extend atfs-1(et17) lifespan, while adult-only knockdown has no effect. Overall, these results demonstrate that constitutively active ATFS-1 can extend lifespan when expressed at low levels and that this lifespan extension is not dependent on the ability of ATFS-1 to enhance resistance to stress.

developmental biology↗

bioRxiv2023

An interactive web application for exploring human plasma and fibroblast metabolomics data from patients with inborn errors of metabolism

Metabolomic profiling is instrumental in understanding the systemic and cellular impact of inborn errors of metabolism (IEMs), monogenic disorders caused by pathogenic genomic variants in genes involved in metabolism. This study encompasses untargeted metabolomics analysis of plasma from 474 individuals and fibroblasts from 67 subjects, incorporating healthy controls, patients with 65 different monogenic diseases, and numerous undiagnosed cases. We introduce a web application designed for the in-depth exploration of this extensive metabolomics database. The application offers a user-friendly interface for data review, download, and detailed analysis of metabolic deviations linked to IEMs at the level of individual patients or groups of patients with the same diagnosis. It also provides interactive tools for investigating metabolic relationships and offers comparative analyses of plasma and fibroblast profiles. This tool emphasizes the metabolic interplay within and across biological matrices, enriching our understanding of metabolic regulation in health and disease. As a resource, the application provides broad utility in research, offering novel insights into metabolic pathways and their alterations in various disorders.

bioinformatics↗

bioRxiv2023

Electron transport inhibition increases cellular dependence on purine transport and salvage

Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. While many studies have documented the importance of mitochondrial oxidative phosphorylation (OXPHOS) in tumor growth, some cancer cells experience conditions of reduced OXPHOS in vivo and induce alternative metabolic pathways to compensate. To assess how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts and plasma from patients with inborn errors of mitochondrial metabolism, and in cancer cells subjected to inhibition of the electron transport chain (ETC). All these analyses revealed extensive perturbations in purine-related metabolites; in non-small cell lung cancer (NSCLC) cells, ETC blockade led to purine metabolite accumulation arising from a reduced cytosolic NAD+/NADH ratio (NADH reductive stress). Stable isotope tracing demonstrated that ETC deficiency suppressed de novo purine nucleotide synthesis while enhancing purine salvage. Analysis of NSCLC patients infused with [U-13C]glucose revealed that tumors with markers of low oxidative mitochondrial metabolism exhibited high expression of the purine salvage enzyme HPRT1 and abundant levels of the HPRT1 product inosine monophosphate (IMP). ETC blockade also induced production of ribose-5 phosphate (R5P) by the pentose phosphate pathway (PPP) and import of purine nucleobases. Blocking either HPRT1 or nucleoside transporters sensitized cancer cells to ETC inhibition, and overexpressing nucleoside transporters was sufficient to drive growth of NSCLC xenografts. Collectively, this study mechanistically delineates how cells compensate for suppressed purine metabolism in response to ETC blockade, and uncovers a new metabolic vulnerability in tumors experiencing NADH excess.

biochemistry↗

bioRxiv2023

Endosomal trafficking protein TBC-2 isrequired for the longevity of long-livedmitochondrial mutants

Mutations that result in a mild impairment of mitochondrial function can extend longevity. Previous studies have shown that the increase in lifespan is dependent on stress responsive transcription factors, including DAF-16/FOXO, which exhibits increased nuclear localization in long-lived mitochondrial mutants. We recently found that the localization of DAF-16 within the cell is dependent on the endosomal trafficking protein TBC-2. Based on the important role of DAF-16 in both longevity and resistance to stress, we examined the effect of disrupting tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants nuo-6 and isp-1 in C. elegans. Loss of tbc-2 markedly reduced the long lifespans of both mitochondrial mutants. Disruption of tbc-2 also decreased resistance to specific exogenous stressors in nuo-6 and isp-1 mutants. In contrast, tbc-2 inhibition had no effect on oxidative stress resistance or lifespan in isp-1 worms when DAF-16 is absent suggesting that the effect of tbc-2 on mitochondrial mutant lifespan may be mediated by mislocalization of DAF-16. However, this result is complicated by the fact that deletion of daf-16 markedly decreases both phenotypes in isp-1 worms. Surprisingly, disruption of tbc-2 did not prevent the upregulation of DAF-16 target genes in the long-lived mitochondrial mutants, suggesting the possibility that the effect of tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants is at least partially independent of its effects on DAF-16 localization. Overall, this work demonstrates the importance of endosomal trafficking for the extended longevity and enhanced stress resistance resulting from mild impairment of mitochondrial function.

genetics↗

bioRxiv2022

Npas4-mediated dopaminergic regulation of fear memory states

Amygdala circuitry encodes associations between conditioned stimuli and aversive unconditioned stimuli, and also controls fear expression (Pape and Pare, 2010). However, whether and how irrelevant information for unpaired conditioned stimuli (CS-) is discretely processed, and how it was influenced by stress remain unknown. CS- memory is retrievable immediately after fear conditioning, but then becomes silent after memory consolidation in mice. Synaptic pathway from the lateral to the anterior basal amygdala gates the expression of CS- memory, depending upon Npas4-mediated Drd4 synthesis. The upregulation of Npas4-Drd4 axis, which is precluded by corticosterone, shifts functional states of neural engrams for CS- memory toward silent states and limits its retrievability. In here, we demonstrate the cellular and molecular mechanisms regulating the functional states of neural engrams, which can be switched or maintained, supporting discriminative memory.

neuroscience↗

bioRxiv2020

The hexosamine biosynthesis pathway is a targetable liability in lung cancers with concurrent KRAS and LKB1 mutations.

In non-small cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumor suppressor STK11 encoding the kinase LKB1 result in aggressive tumors prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant (KL) cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analyzed tumor metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards an activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KL mutant tumors. KL cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). GFPT2 inhibition selectively reduced KL cell growth in culture and xenografts. Our results define a new metabolic vulnerability in KL tumors and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

cancer biology↗

bioRxiv2019

METABOLIC DIVERSITY IN HUMAN NON-SMALL CELL LUNG CANCER CELLS

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes including signal transduction and gene expression patterns arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here we quantified over 100 metabolic features, mostly from 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

cancer biology↗

bioRxiv2018

NFAT5 participates in Inducible Nitric Oxide Synthase activation by hypoxia in MEF cells.

We previously described the protective role of NFAT5 during hypoxia, in an independent way of HIF-1. Alternatively, inducible NO synthase (iNOS) is also induced by hypoxia. The aim of this study was to establish the NFAT5 target gene in mouse embryonic fibroblasts (MEF) cell stimulated by hypoxia. NFAT5, iNOS, NO level, aquaporin 1 (AQP1) and urea transporter 1 (UTA1) were induced by low oxygen levels in MEF cells. Additionally, NFAT5 and UTA1 were induced in reoxygenation (after 24hrs of hypoxia). NFAT5 transactivation domain (TAD) was induced during hypoxia and hypoxia/reoxygenation. Two MEF cells line independently produced for altered NFAT5 (Knockout and DBD-mutant) lost the iNOS and AQP1 induction by low oxygen. The iNOS induction was recovered in NFAT5-KO MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not for NFAT5 DBD-mutant MEF cells, explained by its dominant negative effect. Finally, we found a negative feedback loop of iNOS effect over NFAT5 protein abundance. This work provides a relevant information about signaling pathway of NFAT5 during adaptive responses to oxygen depletion.

physiology↗