Biology subjects

Ying, C.

Publications and source records attributed to Ying, C..

11 recordsLinked to original sources

bioRxiv2026

FluoroFate: A generalisable platform for time-resolved single-cell analysis of cell fate enables quantification of cell death dynamics

Fundamental cellular decisions of life and death are governed by intricate and tightly regulated intracellular signalling pathways that determine whether cells proliferate, enter quiescence, or undergo programmed cell death (apoptosis). Live-cell fluorescence imaging enables these processes to be observed in real time at single-cell resolution, but two problems limit their study. First, existing biosensors do not allow apoptotic status and cell cycle progression to be resolved in tandem within the same cell. Second, interpreting live-cell imaging data is challenging even where multiplex reporters exist, as the biological meaning of fluorescent signals depends on their temporal ordering, and large-scale imaging experiments generate complex, multidimensional data that are difficult to analyse systematically and at scale. Here we address both problems. We present FluoroFate, a generalisable and user-friendly graphical interface-driven tool for time-resolved single-cell analysis of multiplex live-cell imaging datasets, which integrates existing, robust deep learning-based segmentation, cell tracking, and temporal classification methods to quantify fluorescent reporter dynamics in individual cells across time without the need for specialist computational expertise. Alongside FluoroFate, we develop tricistronic Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci) and apoptosis biosensors, enabling simultaneous monitoring of cell cycle progression and caspase activation within the same cell. Applying FluoroFate, we resolve apoptotic and non-apoptotic cell death at the single-cell level based on the temporal ordering of Annexin V and propidium iodide signals, identifying distinct kinetic and phenotypic cell death profiles in response to pharmacological perturbation. We highlight divergent temporal dynamics and modes of cell death between birinapant and cycloheximide treatment, reflecting differences in how TNF/TNFR1 signalling is disrupted by these agents. At the single-cell level, we uncover parallel, independently regulated death programmes, demonstrating that loss of RIPK1 selectively impairs apoptotic cell death whilst leaving non-apoptotic death largely unaffected. We then use FluoroFate to analyse timelapse images of our combined Fucci-apoptosis reporters, resolving cell cycle progression and caspase activation within the same cell over time. Together, FluoroFate and our new cell cycle and apoptosis biosensors represent a broadly applicable platform for extracting mechanistic insight from live-cell imaging data.

cell biology↗

bioRxiv2025

Continuous, Low Latency Estimation of the Size and Shape of Single Proteins from Real-Time Nanopore Data

Existing approaches for nanopore sensing typically analyze resistive pulses from the translocation of individual proteins through the nanopore after completing the experiment. This approach foregoes instantaneous protein identification and precludes real-time experimental control. Here, we introduce a method for the analysis of real-time nanopore data capable of characterizing the size and approximate shape of proteins within a millisecond response time during data acquisition. The implemented real-time Two-Sliding Window (TSW) peak detection algorithm makes it possible, for the first time, to filter data, process baselines, and extract resistive pulse information during nanopore recordings using a stream of single data points. This approach achieves a computational throughput of 40 MB/s on a 1.8 GHz laptop CPU. We compared the accuracy of dwell time determination of the TSW algorithm with an established offline threshold searching (TS) algorithm, using simulated resistive pulses. The TSW algorithm accurately extracted events with dwell times greater than 1.5 times the reciprocal of the systems cutoff frequency, 1.5 x (fc)-1. Moreover, the TSW algorithm effectively determined the dwell time of resistive pulses with large intra-event modulation, which is crucial for resolving non-spherical protein-induced fluctuations. Finally, we verify experimentally that integrating the TSW algorithm into the data acquisition process makes it possible to determine the approximate shape and volume of proteins within low-millisecond response times. This real-time data analysis approach potentially provides instant feedback control for manipulating single proteins during or immediately after translocation. This analysis also improves data storage efficiency during recordings with a high sampling rate.

biophysics↗

bioRxiv2025

Direct observation of single intrinsically disordered proteins in solution

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) in structured proteins are integral to many biological processes including neurotransmitter regulation, microtubule regulation, and transcription. IDP/IDRs are heterogenous, existing in a conformational ensemble of various interconnected states without a definitive tertiary structure. The high dynamicity of IDPs/IDRs limits ensemble protein characterisation techniques from capturing their properties, and measurements at the single-molecule level are hampered by the necessity to label the protein or modify its microenvironment, affecting their biophysics. Consequently, our understanding of IDPs/IDRs is limited, translating to a lack of knowledge of their roles in related diseases including Alzheimers disease, Parkinsons disease, and various cancers. This work presents the first experimental observation of unmodified IDP/IDR conformational dynamics in vitro, at the single-molecule level in real time, achieved by trapping individual IDPs/IDRs in a nanoscale volume using nanoaperture optical tweezers. Our results reveal that IDPs/IDRs exhibit significantly larger conformational variations in solution compared to globular proteins of similar size, as expected. We demonstrate that phosphorylation of native tau-441 by glycogen synthase kinase 3-beta (GSK3{beta}-tau) induces compaction and reduced conformational dynamics. We further observed a disorder-to-order transition during binding of an IDR, the N-terminal region of Src-associated protein in mitosis of 68 kDa (Sam68), to G8.5 RNA. The capability of nanoaperture optical tweezers to monitor the dynamic behaviours of single, unmodified IDPs/IDRs provides a powerful approach to advance our understanding of their elusive behaviours and further decode their roles in associated diseases.

biophysics↗

bioRxiv2025

Asymmetric subspecies-selection of an NLR-TF immune module and reconstruction of broad-spectrum disease resistance in rice

Artificial selection has greatly shaped crop agronomic traits; however, the mechanistic basis of immunity selection has remained elusive. This study identifies a new rice NLR XA48 and its downstream transcription factor OsVOZ1, which confer bacterial blight resistance. XA48 perceives an ancient pathogen effector, XopG, to activates effector-triggered immunity (ETI). The XA48-OsVOZ1 module has undergone subspecies-specific selection. Xa48 is retained in indica but functionally lost in japonica rice. OsVOZ1 has also diverged into two haplotypes, indica kept both OsVOZ1A/S alleles that match XA48; while japonica only inherited OsVOZ1A that greatly decreases yield when Xa48 is reintroduced into japonica, mechanistically explaining the Xa48 loss in japonica. We resurrected wild rice broad-spectrum resistance by stacking XA48-mediated ETI with XA21-mediated pattern-triggered immunity (PTI). Thus, our study reveals that the asymmetric selection of an NLR-TF module shapes both disease resistance and reproduction, and provides a paradigm for breeding crops by harnessing the immunity of wild relatives.

genetics↗

bioRxiv2024

Elucidating the kinetic and thermodynamic insight into regulation of glycolysis by lactate dehydrogenase and its impact on tricarboxylic acid cycle and oxidative phosphorylation in cancer cells

Lactate dehydrogenase (LDH) stands at the intersection of pyruvate metabolism. While it is believed that inhibition of LDH redirects pyruvate to mitochondrial metabolism, suppressing glycolysis and boosting oxidative phosphorylation, the mechanism remains largely unexplored. We found that individual LDH A or B knockouts had minimal impact on glycolysis, tricarboxylic acid cycle (TCA cycle), or oxidative phosphorylation (OXPHOS). However, combining LDH knockout with LDH inhibitor GNE-140 significantly suppressed these processes. Inhibition of LDH led to an increase in free NADH concentration and a decrease in free NAD+ concentration, the reduced free NAD+ concentration inhibited GAPDH, disrupting the balance of glycolytic intermediates, which were linked with thermodynamic shift of the Gibbs free energy of reactions between phosphofructokinase 1 (PFK1) and phosphoglycerate mutase (PGAM) in the glycolytic pathway, favoring their reverse direction. This disrupted glycolysis led to impaired TCA cycle and mitochondrial respiration due to reduced pyruvate and glutamine carbon influx into TCA cycle. Under hypoxia, LDH inhibition had a stronger effect, inducing energy crisis, redox imbalance, and cancer cell death. Our study reveals LDHs intricate control over glycolysis, TCA cycle, and mitochondrial respiration, highlighting the interplay of enzyme kinetics and thermodynamics in metabolic pathways, a crucial aspect for understanding metabolic regulation. Impact statementThis study elucidates a biochemical mechanism by which lactate dehydrogenase influences glycolytic flux in cancer cells, revealing a kinetic- thermodynamic interplay that contributes to metabolic regulation.

biochemistry↗

bioRxiv2024

Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects. The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a global resolution of 3.8 and 3.6 [A] respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and further characterized using plasmonic optical tweezers and hydrogen-deuterium exchange mass spectrometry. To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed invasion blocking properties of anti-TcPOP polyclonal response via parasite lysis and its cross-reactivity versus closely-related POPs but not with human homologues. We were also able to produce and characterise three monoclonal antibodies, one of which showed neutralising properties and inhibition of parasite invasion via non-lytic mechanism. Collectively, our findings provide critical structural and functional insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.

biophysics↗

bioRxiv2023

Structural Flexibility and Disassembly Kinetics of Single Ferritins using Optical Nanotweezers

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. Understanding how various chemicals affect the structural behaviour of ferritin is crucial for unravelling the origins of iron-related diseases in living organisms including humans. In particular, the influence of chemicals on ferritins dynamics and iron release is barely explored at the single-protein level. Here, by employing optical nanotweezers using double nanohole (DNH) structures, we examined the effect of ascorbic acid (reducing reagent) and pH on ferritins conformational dynamics. The dynamics of ferritin increased as the concentration of ascorbic acid approached saturation. At pH 2.0 ferritin exhibited significant structural fluctuations and eventually underwent a stepwise disassembly into fragments. This work, for the first time, tracked the disassembly pathway and kinetics of single ferritins in solution. We identified four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer, and dimer subunits. Moreover, we presented the first single-molecule evidence of the cooperative disassembly of ferritin. Interrogating ferritins structural change in response to different chemicals holds importance for understanding their roles in iron metabolism, hence facilitating further development of medical treatments for the associated diseases.

biophysics↗

bioRxiv2023

Simultaneous Determination of Size and Shape of Single alpha-Synuclein Oligomers in Solution

Soluble oligomers of amyloid-forming proteins are implicated as toxic species in the context of several neurodegenerative diseases. Since the size and shape of these oligomers influences their toxicity, their biophysical characterization is essential for a better understanding of the structure-toxicity relationship. Amyloid oligomers are difficult to characterize by conventional approaches due to their heterogeneity in size and shape, their dynamic aggregation process, and their low abundance. This paper demonstrates that resistive-pulse measurements using polymer-coated solid-state nanopores enable single-particle level characterization of the size and shape of individual Syn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals that nanopore-based characterization agrees well with both methods, while providing better size resolution and elucidating that Syn samples are composed of stable oligomer sub-populations that contain multiples of approximately 12 monomers (i.e., 12-, 24-, 48-, 60-, 84-mers). Applying the unique capability of nanopores to approximate particle size and shape to picomolar concentrations of Syn oligomers in the putatively toxic size range, revealed shapes that agree well with previous estimates by cryo-EM with the added advantage that nanopore-based analysis occurs rapidly, in solution, and has the potential to become a widely accessible technique.

biophysics↗

bioRxiv2020

Hsp22 is the key sensor and balancer in mitochondrial dynamic associated metabolic reprogramming

Altered mitochondrial dynamics are commonly seen in tumors and Parkinsons disease (PD), but the exact mechanism is unclear. We observed in tumor and PD flies, interference of mitochondrial fission or fusion reverses metabolic reprogramming and attenuates pathogenic phenotypes, indicating the rebalanced mitochondrial dynamics can re-establish cellular homeostasis. Surprisingly, we found Hsp22 is highly induced in tumor and PD flies. Hence, Hsp22 overexpression, not only suppressed RAS tumor flies, hepatocellular carcinoma mice, but also rescued PINK1B9 PD flies associating with restored mitochondrial homeostasis and reversing metabolic reprogramming. We speculate that mitochondrial dynamics is normally in a constant dynamic equilibrium, as if on a continually oscillating seesaw, tumor and PD are like the two extreme states of the seesaw which can be corrected by the promising target, Hsp22. One Sentence SummaryMitochondrial dynamics defects including tumor and PD lead to metabolic reprogramming accompanied with Hsp22 induction, vice versa, overexpression of Hsp22 reverses the mitochondrial dynamic dysfunction caused pathogenic metabolism.

cancer biology↗

bioRxiv2019

MACF1 Facilitates SMAD7 Nuclear Translocation to Drive Bone Formation in Mice

MACF1 is a large crosslinker that contributes to cytoskeleton integrity and cell differentiation. Loss of MACF1 impairs multiple cellular functions in neuron development and epidermal migration, and is the molecular basis for many diseases such as heart failure and Parkinsons disease. MACF1 is highly abundant in bones, however, its involvements in osteogenic differentiation and bone formation are still unknown. In this study, by conditional gene targeting to delete the Macf1 gene specifically in MSCs, we observed ossification retardation and bone loss in MACF1 deficient mice in different developmental stages, which we traced to disorganized cytoskeleton and decreased osteogenic differentiation capability in MSCs. Further, we show that MACF1 interacts and facilitates SMAD7 nuclear translocation to initiate downstream transcription. These findings are hopefully to expand the biological scope of MACF1 in bones, and provide experimental basis for targeting MACF1 in degenerative bone diseases such as osteoporosis.

developmental biology↗

bioRxiv2018

Identification and quantification of Lyme pathogen strains by deep sequencing of outer surface protein C (ospC) amplicons

Mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for diagnosis, treatment, and surveillance of Lyme disease. Here we describe a novel protocol for differentiating Lyme strains based on deep sequencing of the hypervariable outer-surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high-throughput, quantitative, and able to detect new pathogen strains. We applied the method to over one hundred infected Ixodes scapularis ticks collected from New York State, USA in 2015 and 2016. Analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among co-infecting strains, and presence of a new strain closely related to Borreliella bissettiae. A supporting bioinformatics pipeline has been developed. With the newly designed pair of universal ospC primers targeting intergenic sequences conserved among all known Lyme pathogens, the protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and clinical specimens across the globe.

microbiology↗