Biology subjects

Liu, Y.-T.

Publications and source records attributed to Liu, Y.-T..

11 recordsLinked to original sources

bioRxiv2026

De novo design of CR2 binder as vaccine scaffold

Efficient B cell activation during vaccine-induced humoral immunity relies on both B cell receptor (BCR) antigen recognition and synergistic signaling from co-receptors. Complement receptor 2 (CR2), the primary BCR co-receptor on B cells, lowers the activation threshold and amplifies downstream kinase signaling by orders of magnitude when engaged by complement fragment C3d decorated antigens. Targeting CR2 therefore represents a rational vaccine enhancement strategy, yet native C3d suffers from low affinity, poor stability, and manufacturing challenges. Here, we report the de novo design of a highly stable, high-affinity CR2 binder using deep learning driving protein design methods. Biophysical characterization, high-resolution cryoEM structural determination, and functional assays in vitro and in vivo confirm that the designed binder matches computational design models and specifically engages CR2 to boost B cell activation. When fused to antigen as a vaccine scaffold, the trimeric CR2 binder elicits robust humoral immune responses comparable to nanoparticle vaccines, while retaining the simplicity of single-chain protein production. Our work establishes a modular CR2 targeting vaccine scaffold platform with broad translational potential for next-generation protein vaccines.

immunology↗

bioRxiv2026

Using spIsoNet to address the preferred-orientation problem in cryoEM reconstructions

Cryogenic electron microscopy (cryoEM) is now routinely used for high-resolution structure determination of biological macromolecules. However, many biological specimens exhibit varying degrees of preferred orientation on cryoEM grids, resulting in uneven sampling of three-dimensional Fourier space. This orientation bias produces anisotropic reconstruction artifacts and, in severe cases, can exacerbate particle misalignment during iterative refinement, thereby limiting the success rate of near-atomic resolution cryoEM structure determination. This protocol provides a practical guide for applying spIsoNet, a self-supervised deep-learning method, to mitigate preferred-orientation issues in cryoEM reconstructions. We describe two complementary workflows: (1) map Anisotropy Correction to correct anisotropic artifacts of cryoEM maps and (2) particle Misalignment Correction, which integrates spIsoNet with RELION external reconstruction to improve particle-pose estimation. We demonstrate these workflows using two influenza hemagglutinin (HA) trimer datasets representing moderate and severe degrees of preferred-orientation bias. The protocol includes installation instructions, parameter-selection guidance, quality-control checkpoints and troubleshooting advice, and can typically be completed in [~]7 hours on a workstation equipped with four NVIDIA A100 GPUs. Together, these workflows provide step-by-step guidance for using the open-source spIsoNet software to mitigate the preferred-orientation problem directly from experimental data.

biophysics↗

bioRxiv2025

Scalable Non-negative Matrix Factorization of the Human Cell Census Reveals Interpretable Transcriptional Programs

Large single-cell atlases now span tens of millions of cells, yet few provide reusable and interpretable reference representations that support direct biological reasoning at atlas-scale. Here, we present an interpretable Non-negative Matrix Factorization reference embedding of 28.5 million healthy cells and approximately 60,000 genes from the Human Cell Census. The resulting gene and cell weights define additive transcriptional programs that align with annotated cell types and organized biological pathways. New datasets can be projected into this fixed reference space without fine-tuning or retraining, as we demonstrate using an independent cystic fibrosis dataset. This resource provides a transparent coordinate system for exploratory analysis and hypothesis generation, complementing deep embeddings that prioritize integration or prediction with a representation designed for interpretability and reuse.

bioinformatics↗

bioRxiv2025

Transcriptome-wide Identification of GlycoRNAs by Clier-seq Pipeline

RNA molecules can undergo modification by N-glycans and be displayed on the cell surface. However, recent studies have focused primarily on N-glycan modifications on small RNAs less than 200 nt in length; the transcriptome-wide subtypes of glycosylated RNAs (glycoRNAs) remain poorly characterized. A validation system, which represents only a fraction of the total transcripts, has yet to be established to analyze glycoRNAs accurately. In this study, we aimed to comprehensively characterize transcriptome-wide global glycoRNAs and novel glycoRNA subtypes in both epithelial cells and B cells. Using metabolic labeling and density gradient centrifugation methods, we identified glycoRNAs predominantly below 2000 nt in both epithelial cells and B cells. We then developed the Clier-seq (Click chemistry-based enrichment of glycoRNAs sequencing) method to maximize the coverage of glycoRNAs (ranging from 50 to 2000 nt) and utilized the HISAT-StringTie-Ballgown pipeline to predict novel glycoRNA subtypes. We also established Clier-qPCR assays (click chemistry-based enrichment of glycoRNAs RT-qPCR) to validate the specificity of the candidate glycoRNAs. We demonstrated that transfer RNAs (tRNAs), particularly tRNAs (Ser), tRNAs (Thr), tRNAs (Val), and tRNAs (lys), are the primary targets of glycosylation. Additionally, we found that vault RNAs (vtRNAs), specifically vtRNA2-1, are glycosylated. Furthermore, we discovered several novel glycosylated long noncoding RNAs ranging from 200-400 nt in length. Herein, we propose a standardized bioinformatics pipeline for glycoRNA research, enabling accurate and comprehensive identification of glycoRNAs throughout the transcriptome.

molecular biology↗

bioRxiv2022

Discovery, Structure, and Function of Filamentous 3-Methylcrotonyl-CoA Carboxylase

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments and determined its structure at near-atomic resolution using cryoEM. 6{beta}6 LtMCC dodecamers assemble in a twisted-stacks architecture, manifesting as supramolecular rods extending up to approximately 400 nanometers. LtMCCs in filaments bind biotin but are not covalently biotinylated and lack coenzyme A. Filaments elongate by stacking 6{beta}6 LtMCCs onto the exterior -trimer of the terminal 6{beta}6 dodecamer. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state within the mitochondrial matrix. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the functional significance of polymerization in the carboxylase superfamily and beyond.

molecular biology↗

bioRxiv2022

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and lipid biosynthesis capacity

Mitochondria are often considered the power stations of the cell, playing critical roles in various biological processes such as cellular respiration, photosynthesis, stress responses and programmed cell death. To maintain the structural and functional integrities of mitochondria, it is crucial to achieve a defined membrane lipid composition between different lipid classes wherein specific proportions of individual lipid species are present. Although mitochondria are capable of self-synthesizing a few lipid classes, many phospholipids are synthesized in the endoplasmic reticulum and transferred to mitochondria via membrane contact sites, as mitochondria are excluded from the vesicular transportation pathway. However, knowledge on the capability of lipid biosynthesis in mitochondria and the precise mechanism of maintaining the homeostasis of mitochondrial lipids is still scarce. Here we describe the lipidome of mitochondria isolated from Arabidopsis leaves, including the molecular species of glycerolipids, sphingolipids and sterols to depict the lipid landscape of mitochondrial membranes. In addition, we define proteins involved in lipid metabolism by proteomic analysis and compare our data with mitochondria from cell cultures since they still serve as model system. Proteins putatively localized to the membrane contact sites are proposed based on the proteomic results and online databases. Collectively, our results suggest that leaf mitochondria are capable - with the assistance of membrane contact site-localised proteins - of generating several lipid classes including phosphatidylethanolamines, cardiolipins, diacylgalactosylglycerols and free sterols. We anticipate our work to be a foundation to further investigate the functional roles of lipids and their involvement in biochemical reactions in plant mitochondria. One sentence summaryThe lipid landscape of plant mitochondria suggests that they are capable in generating several phospholipid classes with the assistant of membrane contact site-localized proteins.

plant biology↗

bioRxiv2022

Optogenetic Manipulation of Cell Migration with High Spatiotemporal Resolution Using Lattice Lightsheet Microscopy

Lattice lightsheet microscopy (LLSM) is modified with the aim of manipulating cellular behavior with subcellular resolution through three-dimensional (3D) optogenetic activation. In this study, we report a straightforward implementation of the activation source in LLSM in which the stimulating light can be generated by changing the spatial light modulator (SLM) patterns and the annual masks. As a result, a Bessel beam as a stimulation source is integrated into the LLSM without changing the optical configuration, achieving better spatiotemporal control of photoactivation. We show that the energy power required for optogenetic reactions is lower than 1 nW (or 24 mW/cm2), and membrane ruffling can be activated at different locations within a cell with subcellular resolution. We also demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable damage to cells.

cell biology↗

bioRxiv2021

Isotropic Reconstruction of Electron Tomograms with Deep Learning

Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic "missing-wedge" problem has presented major challenges in visualization and interpretation of tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs the missing-wedge information and increases signal-to-noise ratio, using the knowledge learned from raw tomograms. Without the need for sub-tomogram averaging, Isonet generates tomograms with significantly reduced resolution anisotropy. Applications of IsoNet to three representative types of cryoET data demonstrate greatly improved structural interpretability: resolving lattice defects in immature HIV particles, establishing architecture of the paraflagellar rod in Eukaryotic flagella, and identifying heptagon-containing clathrin cages inside a neuronal synapse of cultured cells. Therefore, by overcoming two fundamental limitations of cryoET, IsoNet enables functional interpretation of cellular tomograms without sub-tomogram averaging. Its application to high-resolution cellular tomograms should also help identify differently oriented complexes of the same kind for near-atomic resolution sub-tomogram averaging.

cell biology↗

bioRxiv2020

Mesophasic organization of GABAA receptors in hippocampal inhibitory synapse

Information processing in the brain depends on synaptic transmission and plasticity, which in turn require specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density (PSD). However, how these molecules are organized in situ remains largely elusive, limiting our mechanistic understanding of synaptic formation and functions. Here, we have developed template-free classification of over-sampled sub-tomograms to analyze cryo-electron tomograms of hippocampal synapses, enabling us to identify type-A {gamma}-aminobutyric acid receptor (GABAAR) in inhibitory synapses and determine its in situ structure at 19 [A] resolution. We found that these receptors are organized hierarchically: from GABAAR super-complexes with a fixed 11-nm inter-receptor distance but variable relative angles, through semi-ordered two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. This assembly aligns with condensates of postsynaptic scaffolding proteins and putative presynaptic vesicle release sites. Such mesophasic self-organization may allow synapses to achieve a "Goldilocks" state with a delicate balance between stability and flexibility, enabling both reliability and plasticity in information processing.

neuroscience↗

bioRxiv2019

Loss of CHCHD2 and CHCHD10 activates OMA1 peptidase to disrupt mitochondrial cristae phenocopying patient mutations in vivo

Dominant mutations in the mitochondrial paralogs CHCHD2 (C2) and CHCHD10 (C10) were recently identified as causing Parkinsons disease and ALS/FTD/myopathy, respectively. Disruption of mitochondrial cristae has been observed in mutant C10 patient tissues and animal models, but the mechanism for this disruption remains controversial. Additionally, C10 patient mutant knock-in (KI) mice were recently reported to activate a mitochondrial integrated stress response (mt-ISR) and develop cardiomyopathy not seen in C10 knockout (KO) mice, calling into question whether mutant C10 pathogenesis is related to C2/C10 normal function or purely toxic gain of function. Here, using the first C2/10 double knockout (DKO) mice, we report that C10 pathogenesis and the normal function of C2/10 are intimately linked. Similar to patients with C10 mutations, we found that C2/10 DKO mice (but not either single KO mice) have disrupted mitochondrial cristae, due to cleavage of the mitochondrial shaping protein L-OPA1 by the stress-induced peptidase OMA1. OMA1 was found to be activated similarly in affected tissues of mutant C10 KI mice, demonstrating that L-OPA1 cleavage is a novel mechanism for cristae abnormalities due to both C10 mutation and C2/C10 loss, and that OMA1, a driver of neurodegeneration in other contexts, may be a therapeutic target. Finally, C2/10 DKO mice partially phenocopied mutant C10 KI mice with the development of cardiomyopathy and activation of the mt-ISR in affected tissues, tying mutant C10 pathogenesis to C2/C10 function.

cell biology↗

bioRxiv2018

Visualization of axonal protein allocation in Drosophila with whole brain localization microscopy

Long-term memory (LTM) formation requires learning-induced protein synthesis in specific neurons and synapses within a neural circuit. Precisely how neural activity allocates new proteins to specific synaptic ensembles, however, remains unknown. We developed a deep-tissue super-resolution imaging tool suitable for single-molecule localization in intact adult Drosophila brain, and focused on the axonal protein allocation in mushroom body (MB), a central neuronal structure involved in olfactory memory formation. We found that insufficient training suppresses LTM formation by inducing the synthesis of vesicular monoamine transporter (VMAT) proteins within a dorsal paired medial (DPM) neuron, which innervates all axonal lobes of the MB. Surprisingly, using our localization microscopy, we found that these learning-induced proteins are distributed only in a subset of DPM axons in specific sectors along the MB lobes. This neural architecture suggests that sector-specific modulation of neural activity from MB neurons gates consolidation of early transient memory into LTM.

neuroscience↗