Biology subjects

Diao, J.

Publications and source records attributed to Diao, J..

At least 19 recordsLinked to original sources

bioRxiv2025

Multicolored, Sonosensitizer-optimized Organic Mechanoluminescent Nanoparticles for Functional Sono-optogenetics

Light enables precise visualization and control of cellular processes, but its utility in deep tissues is fundamentally limited by poor optical penetration, particularly in the deep brain. Ultrasound-triggered mechanoluminescence offers a non-invasive strategy for remote light delivery, yet existing organic systems remain monochromatic and low-intensity, largely due to an incomplete understanding of ultrasound-induced emission. Here, we report a multicolor mechanoluminescence platform that couples reactive oxygen species-responsive chemiluminescent donors with fluorescent acceptors via Forster resonance energy transfer, generating tunable emission from blue (459 nm) to red (592 nm). Systematic screening potentially reveals that electronic energy gap-dependent reactive oxygen species generation serves as a predictive design principle for high-performance mechanoluminescent materials. The emitted spectrum and intensity are sufficient to activate ChR2 and ChRmine and inhibit eOPN3, enabling bidirectional, fiber-free neuromodulation under focused ultrasound. By integrating spatially precise ultrasound with programmable photon output, this platform establishes a non-invasive strategy for deep-tissue neural monitoring and provides a foundation for applications in bioimaging, gene editing, and precision therapeutics.

bioengineering↗

bioRxiv2025

Modulating inter-mitochondrial contacts to increase membrane potential for mitigating blue light damage

Mitochondrial membrane potential (MMP) is essential for mitochondrial functions, yet current methods for modulating MMP lack precise spatial and temporal control. Here, we present an optogenetic system that enables reversible formation of inter-mitochondrial contacts (mito-contacts) with high spatiotemporal precision. Blue light stimulation induces rapid formation of mito-contacts, which fully dissipate upon cessation of illumination. These light-induced mito-contacts can enhance MMP, leading to increased ATP production under stress conditions. Moreover, in human retinal cells and C. elegans, high MMP induced by mito-contacts alleviates the deleterious effects of prolonged blue light exposure, restoring energy metabolism and extending organismal lifespan. This optogenetic approach provides a powerful tool for modulating MMP and offers potential therapeutic applications for diseases linked to mitochondrial dysfunction.

biophysics↗

bioRxiv2025

A Tonic Signaling Code Predicts CAR-T Cell Efficacy in Diffuse Midline Glioma

Diffuse midline glioma (DIPG/DMG) is a uniformly fatal pediatric brain tumor with no effective cure. Although CAR T-cell therapy shows promise, clinical outcomes remain inconsistent due to limited persistence and premature exhaustion. Reliable predictive biomarkers are lacking, and proposed exhaustion or stemness markers provide limited utility. Here, we systematically compare multiple CAR-T constructs targeting clinically-relevant antigen B7-H3 and identify antigen-independent CAR activation, or tonic signaling, as a key determinant of therapeutic performance. We find that B7-H3 CAR-T cells with restrained tonic signaling display superior tumor killing, persistence, and resistance to exhaustion, along with reduced CAR membrane clustering, in patient-derived DIPG models. Integrated multi-omics and single-cell profiling further reveal a CAR-T tonic signaling-associated gene signature that outperforms conventional exhaustion or stemness markers in predicting therapeutic efficacy across multiple clinical trials, including DIPG and other tumor types. Together, these findings define a mechanistic and predictive framework to guide CAR design and improve clinical outcomes.

cancer biology↗

bioRxiv2025

Cycling Molecular Assemblies for Selective Cancer Cell Golgi Disruption

The Golgi apparatus is a critical organelle responsible for intracellular trafficking and signaling, orchestrating essential processes such as protein and lipid sorting1-5. Dysregulation of its function has been implicated in various pathologies, including obesity, diabetes, and cancer, highlighting its importance as a potential therapeutic target. Despite this, the development of tools to selectively target the Golgi in specific cell types remain a significant unmet challenge in imaging and drug discovery. Golgi-specific enzyme activities, such as those mediated by protein acyltransferases and thioesterases6, offer an untapped opportunity to develop subcellularly localized therapeutics. Current approaches predominantly rely on direct protein binding but lack the necessary cell selectivity7, underscoring the unmet need for innovative strategies to selectively disrupt Golgi function in cancer cells. Here, we report the development of cycling molecular assemblies (CyMA), a novel class of small peptide derivatives (e.g., dipeptides), which exploit the unique enzymatic environment of the Golgi to establish futile cycles of reversible S-acylation. These assemblies selectively accumulate in cancer cell Golgi, interfering with protein S-acylation cycles and disrupting organelle homeostasis. CyMA impair key Golgi functions, including protein trafficking, glycosylation, and secretion, while demonstrating selective sparing hepatocytes and immune cells such as M1 macrophages. This selective activity represents a paradigm shift, utilizing an enzyme switch and leveraging intracellular environment rather than direct protein binding. Unlike conventional approaches, CyMA reduce tumor growth, drug resistance, and metastasis by pleiotropically disrupting Golgi related functions. By demonstrating the potential of futile cycles as a therapeutic strategy8, this study introduces a generalizable method for targeting organelle-specific enzyme activities. These findings not only underscore the therapeutic potential of CyMA in cancer but also pave the way for future applications in other Golgi-associated diseases.

biochemistry↗

bioRxiv2024

Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission

Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, -SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin--SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

neuroscience↗

bioRxiv2024

Identification and characterization of the de novo methyltransferases for eukaryotic N6-methyladenine (6mA)

DNA N6-adenine methylation (6mA) is rediscovered as an important epigenetic mark in eukaryotes. We previously identified the MT-A70 family member AMT1 as the maintenance methyltransferase (MTase), but the identity of the de novo MTase(s) for 6mA establishment remains unidentified. The unicellular model eukaryote Tetrahymena thermophila offers a unique opportunity for studying de novo methylation: during sexual reproduction, the 6mA-free zygotic nucleus develops into 6mA-positive new macronucleus (MAC) marked by genome-wide de novo methylation. Here we identified that TET1 and TET2 are essential for de novo 6mA in the new MAC, but not for maintenance 6mA in the somatic MAC. Knockout of TET1 and/or TET2 not only abolished methylation on most methylatable ApT positions, but also attenuated methylation on the rest. The substantial 6mA decrease in turn altered gene expression pattern and reduced the survival rate of sexual progenies, highlighting the importance of dedicated de novo MTase activity.

molecular biology↗

bioRxiv2024

N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine

Post-translational modifications (PTMs) of -synuclein (-syn) such as acetylation and phosphorylation play important yet distinct roles in regulating -syn conformation, membrane binding, and amyloid aggregation. However, how PTMs regulate -syn function in presynaptic terminals remains unclear. Previously, we reported that -syn clusters synaptic vesicles (SV)1, and neutral phospholipid lysophosphatidylcholine (LPC) can mediate this clustering2. Here, based on our previous findings, we further demonstrate that N-terminal acetylation, which occurs under physiological conditions and is irreversible in mammalian cells, significantly enhances the functional activity of -syn in clustering SVs. Mechanistic studies reveal that this enhancement is caused by the N-acetylation-promoted insertion of -syns N-terminus and increased intermolecular interactions on the LPC-containing membrane. Our work demonstrates that N-acetylation fine-tunes -syn-LPC interaction for mediating -syns function in SV clustering.

biophysics↗

bioRxiv2024

Self-regulation of the DNA N6-adenine methyltransferase AMT1 in the unicellular eukaryote Tetrahymena thermophila

DNA N6-adenine methylation (6mA) is involved in gene transcription as a potential epigenetic mark in eukaryotes. Despite the reported methyltransferase (MTase) for 6mA methylation in several eukaryotes, the regulatory mechanisms that govern the activity of 6mA MTase remain elusive. Here, we exploited the 6mA MTase AMT1 to elucidate its self-regulation in the unicellular eukaryote Tetrahymena thermophila. Firstly, detailed endogenous localization of AMT1 was delineated both in vegetative and sexual stages, revealing a correlation between the 6mA reestablishment in the new MAC and the occurrence of zygotically expressed AMT1. Catalytically inactive AMT1 reduced 6mA level on the AMT1 gene and its expression level, suggesting that AMT1 modulated its own transcription via 6mA. Furthermore, AMT1-dependent 6mA regulated the transcription of its target genes thus affecting the cell fitness, as demonstrated by manipulating the dosage of AMT1 using AMT1-RNAi strains. Our findings unveil a positive feedback loop of transcriptional activation on the AMT1 gene and highlight the crucial role of AMT1-dependent 6mA for gene transcription.

genetics↗

bioRxiv2023

uniLIVER: a Human Liver Cell Atlas for Data-Driven Cellular State Mapping

The liver performs several vital functions such as metabolism, toxin removal and glucose storage through the coordination of various cell types. The cell type compositions and cellular states undergo significant changes in abnormal conditions such as fatty liver, cirrhosis and liver cancer. As the recent breakthrough of the single-cell/single-nucleus RNA-seq (sc/snRNA-seq) techniques, there is a great opportunity to establish a reference cell map of liver at single cell resolution with transcriptome-wise features. In this study, we build a unified liver cell atlas uniLIVER by integrative analyzing a large-scale sc/snRNA-seq data collection of normal human liver with 331,125 cells and 79 samples from 6 datasets. Besides the hierarchical cell type annotations, uniLIVER also proposed a novel data-driven strategy to map any query dataset to the normal reference map by developing a machine learning based framework named LiverCT. Applying LiverCT on the datasets from multiple abnormal conditions (1,867,641 cells and 439 samples from 12 datasets), the alterations of cell type compositions and cellular states were systematically investigated in liver cancer.

bioinformatics↗

bioRxiv2023

Quantifying cell viability through organelle ratiometric probing

Detecting cell viability is crucial in research involving the precancerous discovery of abnormal cells, the evaluation of treatments, and drug toxicity testing. Although conventional methods afford cumulative results regarding cell viability based on a great number of cells, they do not permit investigating cell viability at the single-cell level. In response, we rationally designed and synthesized a fluorescent probe, PCV-1, to visualize cell viability under the super-resolution technology of structured illumination microscopy. Given its sensitivity to mitochondrial membrane potential and affinity to DNA, PCV-1s ability to stain mitochondria and nucleoli was observed in live and dead cells, respectively. During cell injury induced by drug treatment, PCV-1s migration from mitochondria to the nucleolus was dynamically visualized at the single-cell level. By extension, harnessing PCV-1s excellent photostability and signal-to-noise ratio and by comparing the fluorescence intensity of the two organelles, mitochondria and nucleoli, we developed a powerful analytical assay named organelle ratiometric probing (ORP) that we applied to quantitatively analyze and efficiently assess the viability of individual cells, thereby enabling deeper insights into the potential mechanisms of cell death. In ORP analysis with PCV-1, we identified 0.3 as the cutoff point for assessing whether adding a given drug will cause apparent cytotoxicity, which greatly expands the probes applicability. To the best of our knowledge, PCV-1 is the first probe to allow visualizing cell death and cell injury under super-resolution imaging, and our proposed analytical assay using it paves the way for quantifying cell viability at the single-cell level.

cell biology↗

bioRxiv2022

Enhancing Mitochondrial Functions by Optogenetic Clustering

Known as the powerhouses of cells, mitochondria and its dynamics are important for their functions in cells. Herein, an optogenetic method that controlling mitochondria to form the clusters was developed. The plasmid named CRY2PHR-mCherry-Miro1TM was designed for the optogenetic system. The photoactivable protein CRY2PHR was anchored to mitochondria, via the specific organelle-targeting transmembrane domain Miro1TM. Under blue light illumination, CRY2PHR can form the oligomerization, called puncta. With the illuminated time extended, the puncta can interact, and the mitochondria were found to form clustering with reversibility and spatiotemporal controllability. The mitochondrial functions were found to enhance after the formation of optogenetic mitochondrial clusters. This method presented here provides a way to control mitochondrial clustering and raise mitochondrial functions up.

cell biology↗

bioRxiv2022

Super-resolution analyzing spatial organization of lysosomes with an organic fluorescent probe

Lysosomes are multifunctional organelles involved in macromolecule degradation, nutrient sensing and autophagy. Live imaging has revealed lysosome subpopulations with dynamics and characteristic cellular localization. An as-yet unanswered question is whether lysosomes are spatially organized to coordinate and integrate their functions. Combined with super-resolution microscopy, we designed a small organic fluorescent probe, TPAE, that targeted lysosomes with a large Stokes shift. When we analyzed the spatial organization of lysosomes against mitochondria in different cell lines with this probe, we discovered different distance distribution patterns between lysosomes and mitochondria during increased autophagy flux. By using SLC25A46 mutation fibroblasts derived from patients containing highly fused mitochondria with low oxidative phosphorylation, we concluded that unhealthy mitochondria redistributed the subcellular localization of lysosomes, which implies a strong connection between mitochondria and lysosomes.

biophysics↗

bioRxiv2020

Novel inhibitors of E. coli lipoprotein diacylglyceryl transferase are insensitive to resistance caused by lpp deletion

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify the first ever described Lgt inhibitors that potently inhibit Lgt biochemical activity in vitro and are bactericidal against wild-type Acinetobacter baumannii and E. coli strains. Unlike inhibition of other steps in lipoprotein biosynthesis, deletion of the major outer membrane lipoprotein, lpp, is not sufficient to rescue growth after Lgt depletion or provide resistance to Lgt inhibitors. Our data validate Lgt as a novel druggable antibacterial target and suggest that inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of downstream steps of bacterial lipoprotein biosynthesis and transport.

microbiology↗

bioRxiv2020

Double-transmembrane domain reduces fusion rate by increasing lipid-protein mismatch

SNARE is the essential mediator of membrane fusion that highly relies on the molecular structure of SNAREs. For instance, the protein syntaxin-1 involving in neuronal SNAREs, has a single transmembrane domain (sTMD) leading to fast fusion, while the syntaxin 17 has a V-shape double TMDs (dTMDs), taking part in the autophagosome maturation. However, it is not clear how the TMD structure influences the fusion process. Here, we demonstrate that the dTMDs significantly reduce fusion rate compared with the sTMD by using an in vitro reconstitution system. Through theoretical analysis, we reveal that the V-shape dTMDs can significantly increase protein-lipid mismatch, thereby raising the energy barrier of the fusion, and that increasing the number of SNAREs can reduce the energy barrier or protein-lipid mismatch. This study provides a physical-chemical mechanistic understanding of SNARE-regulated membrane fusion.

biophysics↗

bioRxiv2020

Mechanism of duplex unwinding by coronavirus nsp13 helicases

The current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family. O_LIWhen dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed. C_LIO_LIAn unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding. C_LIO_LIComparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13. C_LI

biophysics↗

bioRxiv2019

Binding to m6A RNA promotes YTHDF2-mediated phase separation

As the most abundant modification on mRNA in mammal, N6-Methyladenosine (m6A) has been demonstrated to play important roles in various biological processes including mRNA splicing, translation and degradation. m6A reader proteins have been shown to play central roles in these processes. One of the m6A readers, YTHDF2 is localized to the P granules, which are liquid-like droplets where RNA degradation occurs. How YTHDF2 is localized to P granules is unknown. Here we provide evidence that YTHDF2 forms liquid droplets and phase separate, mediated by its low complexity (LC) domains. Interestingly, the ability to phase separate is robustly stimulated by m6A RNAs in vitro. In vivo, YTHDF2 phase separation may in fact be dependent on m6A RNA and YTHDF2 binding to m6A RNA, since a YTHDF2 m6A-binding defective mutant or a wildtype YTHDF2 assayed in cells lacking m6A RNAs, both fail to phase separate. The ability of phase separate is not limited to YTHDF2; we find other members of the YTH-domain m6A readers can also undergo phase separation. Our findings suggest that m6A RNA induced phase separation of m6A readers may play an important role in their distributions to different phase-separated compartments in cells.

molecular biology↗

bioRxiv2019

Membrane association of VAMP2 SNARE motif in cells and its regulation by different lipid phases of synaptic vesicle membrane

Vesicle associated membrane protein 2 (VAMP2) contains a conserved SNARE motif that forms helix bundles with the homologous motifs of syntaxin-1 and SNAP25 to assemble into a SNARE complex for the exocytosis of synaptic vesicles (SV). Prior to SNARE assembly, the structure of VAMP2 is unclear. Here, using in-cell NMR spectroscopy, we described the dynamic membrane association of VAMP2 SNARE motif in mammalian cells at atomic resolution, and further tracked the intracellular structural changes of VAMP2 upon the lipid environmental changes. The underlying mechanistic basis was then investigated by solution NMR combined with mass-spectrometry-based lipidomic profiling. We analyzed the lipid compositions of lipid-raft and non-raft phases of SV membrane and revealed that VAMP2 configures distinctive conformations in different phases of SV membrane. The phase of cholesterol-rich lipid rafts could largely weaken the association of SNARE motif with SV membrane and thus, facilitate vesicle docking; While in the non-raft phase, the SNARE motif tends to hibernate on SV membrane with minor activity. Our work provides a spatial regulation of different lipid membrane phases to the structure of core SNARE proteins, which deepens our knowledge on the modulation of SNARE machinery.

biophysics↗

bioRxiv2018

Quantitative Analysis of Interactive Behavior of Mitochondria and Lysosomes Using Structured Illumination Microscopy

Super-resolution optical microscopy has extended the spatial resolution of cell biology from the cellular level to the nanoscale, enabling the observation of the interactive behavior of single mitochondria and lysosomes. Quantitative parametrization of interaction between mitochondria and lysosomes under super-resolution optical microscopy, however, is currently unavailable, which has severely limited our understanding of the molecular machinery underlying mitochondrial functionality. Here, we introduce an M-value to quantitatively investigate mitochondria and lysosome contact (MLC) and mitophagy under structured illumination microscopy. We found that the M-value for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 to 1.0. This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes.

cell biology↗