Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 217 records · Page 12Linked to original sources

bioRxiv2026

Mast cell-driven adipocyte lipolysis promotes post-expansive VAT atrophy and ectopic hepatic steatosis

Excessive visceral fat is an essential risk factor of metabolic dysfunction-associated steatotic liver disease (MASLD). Yet, the cellular and molecular mechanisms implicated in the correlation between visceral fat metabolism and hepatic steatosis remain poorly understood. Here we report post-expansive epididymal adipose tissue (EAT) atrophy and hepatic steatosis in mice fed a high-fat diet (HFD) primarily due to elevated adipocyte lipolysis in EAT. Mast cell (MC) accumulation in EAT represents a lipolysis-associated feature. Pharmacological stabilization of MCs suppresses EAT adipocyte lipolysis, and improves EAT atrophy and hepatic steatosis. MC-derived serotonin (5-HT) correlates with visceral adipose tissue (VAT) lipolysis in HFD-fed mice as well as in MASLD patients. Conditional deletion of 5-HT from MCs or its receptor HTR2b on adipocytes demonstrates that MC-derived 5-HT promotes adipocyte lipolysis, EAT atrophy, and hepatic steatosis by binding on adipocyte HTR2b. These results suggest that MCs and MC-derived 5-HT are potential therapeutic targets for obesity-associated MASLD.

cell biology↗

bioRxiv2026

PAR-Driven Condensation Maintains Stalled Replication Fork Stability

Poly(ADP-ribose) (PAR) is a nucleic acid-like heterogeneous polymer in nature. Recently, it was found to engage in liquid-liquid phase separation (LLPS), generating condensates as an emerging class of subcellular structures with pivotal functions in response to stimuli. As a post-translational modification catalyzed by PAR polymerases (PARPs), PAR is known to modulate many key events in cells. However, its involvement in biomolecular condensation remains elusive. Through an imaging-based screening of small molecules with diverse biological activities, we here discovered that PAR undergoes LLPS upon inhibiting proteasome in different types of cells, resulting in co-condensation of PAR with proteasome and ubiquitin chains in nucleus. This unprecedented co-condensation is dependent on PARP2 not PARP1 and requires K6-linked ubiquitylation. PAR is shown for the first time to directly interact with ubiquitin chains. Notably, stalled DNA replication forks arose from proteasome inhibition are co-localized with PAR-proteasome-ubiquitin chain condensates. By attenuating replication and stabilizing stalled replication forks, PAR-proteasome-ubiquitin chain condensates sustain genomic integrity under proteasomal stress. This work demonstrates a self-protective mechanism in stressed cells and provides fundamental understanding of PAR condensation in cell biology.

biochemistry↗

bioRxiv2026

Phosphorylation Protects Oncogenic RAS from LZTR1-Mediated Degradation

Oncogenic KRAS and NRAS mutations are common in hematologic malignancies, but how they signal is less well characterized than in carcinomas. To uncover novel RAS biology and potential therapeutic vulnerabilities, we employed a multi-omics screening approach in multiple myeloma to identify regulators of RAS activity. We report that the phosphatase PP1C dephosphorylates the conserved T148 residue on RAS, which in turn permits LZTR1-dependent proteasomal degradation. Notably, LZTR1 is ineffective against KRAS A146 gain-of-function mutations, which are adjacent to T148 and prevalent in hematologic cancers. Remarkably, we find that KRAS protein is four-fold less stable in hematologic versus carcinoma cells, offering a unique therapeutic opportunity targeting RAS protein stability mechanisms. The kinases PAK1 and PAK2 shield RAS from LZTR1-dependent degradation by phosphorylating T148, and targeting PAK1/2 activity improves RAS-directed therapy. Collectively, our findings reveal a novel regulatory circuit governing RAS stability that is preferentially active in blood cancers and potentially druggable.

cancer biology↗

bioRxiv2026

Microglia stabilize sleep homeostasis via adenosine A3 receptor signaling

Sleep homeostasis maintains the sleep-wake balance through sleep pressure, a process orchestrated by the accumulation of extracellular adenosine (eADO). Microglial Ca{superscript 2} activity has been implicated in sleep regulation, but the mechanism whereby microglia sense sleep pressure remains unclear. Here we show that microglia regulate sleep homeostasis through brain state-dependent Ca2+ activity driven by adenosine A3 receptor (A3R) signaling. Using miniaturized two-photon microscopy (mTPM) in freely behaving mice, we demonstrate that microglial Ca{superscript 2} activity is rapidly altered by brain-state transitions. Pharmacological experiments reveal that microglial Ca2+ dynamics are predominantly mediated by A3R in response to brain state-dependent eADO oscillations. Microglia-specific deletion of A3R attenuates these state-dependent Ca2+ dynamics, impairs microglial morphological plasticity across sleep-wake cycles, and leads to sleep fragmentation by increasing transitions between wakefulness and non-rapid eye movement (NREM) sleep. Together, these findings establish that microglia regulate sleep homeostasis by stabilizing both wakefulness and NREM sleep, a process critically dependent on eADO-A3R signaling.

neuroscience↗

bioRxiv2026

Multiplexed measurements of protein-protein interactions and protein abundance across cellular conditions using Prod&PQ-seq

Methods to profile protein-protein interactions (PPIs) have limited scalability and can only study a handful of conditions and/or targets. Here, we introduce Prod&PQ-seq, a framework for multiplexed detection and quantification of PPIs and proteins. Our framework uses cross-linked cells, antibody-oligonucleotide conjugates (ab-oligos), and captures PPIs by the DNA-caliper, a specialized oligonucleotide for bidirectional priming of proximal ab-oligos. We benchmarked Prod&PQ-seq using recombinant complexes, titrations and cell mixture experiments and show that our framework is quantitative, reproducible, sensitive and specific. Applying Prod&PQ-seq to study Polycomb Repressive Complex 2 (PRC2) shows that EZH2 inhibition and expression of the oncohistone H3.3K27M weakens both PRC2-H3K27me3 interactions and PPIs within PRC2. Further, H3.1K27M and H3.3K27M variants lead to distinct PPI profiles such as the intensity of H3K27ac-K27M or H3K27ac-EED. Together, Prod&PQ-seq enables detection of changes in PPI composition and intensity and protein quantification across biological conditions, small molecule inhibition and genetic perturbations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/697286v2_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1ed0805org.highwire.dtl.DTLVardef@a9c0d9org.highwire.dtl.DTLVardef@b411fborg.highwire.dtl.DTLVardef@8a12e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Multi-omics characterization of IDH-mutant astrocytoma-derived cell lines reveals NOTCH-regulated plastic quiescent astrocyte-like state

Diffuse IDH-mutant astrocytomas are brain tumors typically diagnosed as low-grade but capable of progressing to higher grades. They exhibit three cellular states resembling astrocytes, oligodendrocytes, and neural progenitor (NPC) cells. Understanding their biology has been challenging due to the lack of relevant in vitro models. Here we established and extensively characterized four astrocytoma cell lines (LGG275, LGG336, LGG85, LGG349) derived from IDH-mutant astrocytoma at different grades, cultured in defined media and analyzed by multi-omics. These lines display growth rates in vitro and in vivo consistent with tumor grade and recapitulate key molecular alterations observed in patient tumors, including IDH1, ATRX, and TP53 mutations, activation of the alternative lengthening of telomeres (ALT) pathway and, in the most aggressive line, amplification of MET and PDGFRA. Single-cell RNA sequencing showed that the 4 astrocytoma lines maintain the three major cellular states observed in patient tumors. A hallmark of higher-grade-derived lines (LGG85, LGG349) is the persistence of NPC-like populations without growth factors, reflecting tumor progression. The LGG275 line most accurately mirrors slow-growing astrocytomas. Using CD44 and GLAST, we isolated astrocyte-like (CD44+/GLAST+) cells from LGG275 that preferentially adopt a quiescent state yet retain remarkable plasticity, generating oligodendrocyte-like cells (CD44-/GLAST-). Transcriptomic and proteomic analyses revealed that astrocyte-like and oligodendrocyte-like cells populations resemble quiescent and activated neural stem (NSC) cells from the adult subventricular zone (SVZ). Finally, we found that NOTCH signaling regulates the balance between astrocytic and oligodendrocytic states, while DLL3, expressed by oligodendrocyte-like cells, modulates both proliferation and phenotype. These cell lines represent valuable resources for dissecting lineage dynamics, heterogeneity, and progression mechanisms in IDH-mutant astrocytomas. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/696808v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@953108org.highwire.dtl.DTLVardef@c6234aorg.highwire.dtl.DTLVardef@3703fcorg.highwire.dtl.DTLVardef@1e6c3a9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LIWe constituted a richly annotated biobank derived from 4 astrocytomas, showing similar characteristics to those found in patients, providing valuable tools to investigate cellular heterogeneity and plasticity, and link with tumor progression. C_LIO_LIscRNA-sequencing revealed three cell states (oligodendrocyte-like, astrocyte-like, and stem cell-like cells) akin to those found in tumors. C_LIO_LIAstrocyte-like cells are quiescent cells, plastic and similar to quiescent neural stem cell (qNSC) from the sub-ventricular zone (SVZ), while oligodendrocyte-like cells are similar to active NSC (aNSC). C_LIO_LIThe Notch pathway plays a role in cell plasticity, enabling a shift towards an astrocyte-like state. C_LI

cancer biology↗

bioRxiv2025

The Hippo Pathway Regulates Corneal Endothelial Regeneration

Corneal endothelial cells (CECs) have limited regeneration capacity in primates while display a potent restorative capability in some species such as rodents after wounding. The mechanism which determines the regenerative capability of CECs remains poorly understood. Here, we identify that the Hippo signaling pathway is inhibited and the downstream effector YAP is activated during CEC wound healing in rabbit and mouse. Knockdown of Yap1 and pharmacological inhibition of YAP suppresses CECs wound healing in rodents in both in vitro and in vivo models. XMU-MP-1, a specific small molecular inhibitor for Hippo signaling pathway, can promote the corneal endothelial regeneration in rodents and corneal endothelial proliferation in cultured primate CECs, as well as in vivo non-human primate CEC wounding model. These findings suggest that Hippo pathway serves as a conserved signal to regulate corneal endothelial regeneration among various species, providing a novel target for non-invasive treatment of corneal endothelium decompensation.

cell biology↗

bioRxiv2025

Sloppiness and Action Confinement in Cell State Transitions: Are Single Cells Sloppy?

Single-cell dynamics during cell state transitions (CST) are highly constrained, enabling precise control. However, there are challenges in achieving a system-level understanding or extracting general principles for CST dynamics due to the complexity of gene-gene interactions. Here, we introduce a new perspective to deal with these challenges using Fisher information. We found that, during CST, single cells exhibit pronounced sloppiness: cell states are sensitive only to a few "stiff" parameters while remaining robust to changes in numerous "sloppy" parameters. Critical transitions coincided with changes in stiff parameters. Moreover, stiff parameters typically exhibited minimal fluctuations and low velocities, whereas sloppy parameters allowed greater flexibility. Together, these findings can be summarized by stating that transition paths approximately adhere to a principle of least action. By characterizing the low dimensionality and constraints of CST through sloppiness and action, our work thus introduces a new conceptual and computational framework for analyzing single-cell dynamics. TeaserExcept when transitions occur, single-cell dynamics exhibit sloppiness, adhering approximately to a least action principle.

biophysics↗

bioRxiv2025

Ferritin Nanoparticle-Based Indirect ELISA for Immunodominant Region Screening and B-cell Epitope Validation of African Swine Fever Virus

African swine fever virus (ASFV) poses a severe threat to the global pig industry, creating an urgent need for effective vaccines and reliable diagnostic tools. While numerous ASFV B-cell epitopes have been reported based on murine-derived monoclonal antibodies (mAbs), such epitopes may not fully represent the antibody targets recognized in naturally infected pigs. Therefore, we need an approach to screen immunodominant regions by evaluating the reactivity of pre-defined epitopes with ASFV-infected pigs sera. In this study, we developed an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant epitope-ferritin nanoparticles (EFNs), as a platform for immunodominant region screening and epitope validation. This assay, hereafter referred to as EFNs-iELISA, shows an approximately twofold improvement in sensitivity compared with synthetic peptide-based ELISA for linear B-cell epitopes, and improved discrimination over GST-His-peptide and BSA-conjugated peptide formats. Using EFNs-iELISA, we evaluated 22 known murine-derived B-cell epitopes and 17 predicted epitopes from ASFV proteins CD2v, P30, P54, and P72 with ASFV-positive and negative porcine sera. According to the calculated cut-off threshold, tested epitopes were classified into different groups with positive or negative reactivity. Our analysis confirmed that among 22 known murine-derived ASFV B-cell epitopes, 16 (72%) also showed positive reactions with porcine-derived sera. Whats more, we also screened 7 new porcine-specific immunoreactive regions on these proteins. The EFNs-iELISA method provides a practical tool for screening immunodominant regions of ASFV, with potential applications in vaccine and diagnostic research.

immunology↗

bioRxiv2025

A New Paradigm of Transcriptional Regulation by the SufR-Like Iron-Sulfur Transcription Factors

SufR is an iron-sulfur ([4Fe-4S]) cluster-containing transcription factor belonging to an uncharacterized domain family (COG2345). It has been shown to negatively regulate the sulfur utilization factor (SUF) Fe-S biogenesis system in several Gram-negative and Gram-positive bacteria including Cyanobacteria, Mycobacteria and Streptomyces. The structural basis for its DNA recognition and transcriptional regulation by the SufR-like proteins remains enigmatic. In this study, we present the cryo-EM structure of Mycobacterium tuberculosis SufR bound to its promoter, revealing a new domain architecture. Our structural, biochemical and molecular analyses show that SufR possesses an unusual [4Fe-4S] cluster coordination environment in the sensory domain and recognizes its promoter via a dual-module mechanism using both the AT-hook and the helix-turn-helix (HTH) DNA-binding motif in the DNA-binding domain. This DNA recognition strategy differs from a canonical winged HTH transcription factor. Moreover, our bioinformatic analysis and structural modeling suggest that SufR and SufR-like proteins in the COG2345 family represent a large, previously uncharacterized family of transcription factors widely distributed across bacteria and archaea. Together, these findings establish SufR-like proteins as a new model for transcriptional regulation by Fe-S transcription factors in prokaryotes and uncover the underappreciated evolutionary versatility of AT-hooks.

biochemistry↗

bioRxiv2025

Multimodal Data Fusion Reveals Morpho-Genetic Variations in Human Cortical Neurons Associated with Tumor Infiltration

We introduce LetsACT (Light-Electron-Transcriptome synergistic ACTomography), a multimodal integration platform that overcomes the limitations of single-modality data acquisition and analysis of human brain cells while synergistically leveraging the strengths of each modality. Our approach enables the rapid sample preparation, cell injection, imaging, and multimodal integration of large-scale human neuronal datasets at single-cell resolution. By generating initial laser-scanning-microscopy based optical reconstruction of neuron morphologies followed by refining them using electron-microscopy derived morphological priors, we have assembled one of the largest human cortical morphology datasets to date: 8,398 neurons from 58 donors, with high cortical coverage. This platform is then applied to studying morphological impact of tumor infiltration. Pyramidal neurons in glioma-infiltrated tissues display clear volume shrinkage in somas and branches, tapering from the soma to nearby dendritic compartments. By integrating these morphological variations with spatial and bulk transcriptomic profiles, we find that glioblastoma tissues exhibit dysregulation of 15.29% of genes, including overexpression of TERT, whereas infiltrated tissues show 7.74% gene dysregulation, characterized by overexpression of tumor suppressors such as CDKN2A and TP53. Our analysis implies that pyramidal neurons observed in these infiltrated tissues may involve an active defense instead of undergoing passive apoptosis. Our finding also indicates that LetsACT establishes a valuable resource for the large-scale, comprehensive morpho-genetic analysis of human tissues.

neuroscience↗

bioRxiv2025

Temporal boundary gating of auditory sensitivity

Perception unfolds over time, but whether continuous sounds are sampled with uniform sensitivity is unknown. We combine human psychophysics, EEG and rodent neurophysiology to show that local auditory change detection is strongly gated by stimulus boundaries. In humans, brief perturbations inserted at different temporal positions within 0.5-1-s tones revealed an inverted U-shaped sensitivity profile: detection was attenuated near sound onset and offset and maximal mid-epoch, with EEG change responses showing a closely matching dependence on change timing. Electrocorticography in awake rats exhibited homologous temporal weighting, and analogous profiles for amplitude changes and visual motion demonstrated cross-feature and cross-modal generality. To uncover circuit mechanisms, we recorded single units along the inferior colliculus-medial geniculate body-auditory cortex pathway together with laminar local field potentials in A1. Onset-locked suppression of change responses emerged in midbrain and was progressively amplified in thalamus and cortex, whereas the full start-end profile was expressed in granular-layer alpha-gamma power. A simple biophysically grounded model in which onset responses saturate and cortical populations integrate over finite temporal windows recapitulates this pattern, explaining how stimulus boundaries disrupt prospective and retrospective integration and thereby degrade change detection near the beginning and end of sounds.

neuroscience↗

bioRxiv2025

A single catalytic domain residue regulates the substrate specificity of a mucin-type O-glycosyltransferase in salivary gland function

Human GalNAc-T1 is a ubiquitously expressed mucin-type O-glycosyltransferase that modulates various biological pathways, including immunity, extracellular matrix formation, and salivary gland development and function. However, substrates of GalNAc-T1 are mostly unknown and the mechanistic details of regulation are not well characterized. Here, we investigate the role of the Drosophila melanogaster GalNAc-T1 ortholog PGANT5 in salivary glands and provide structural and biochemical details into its mechanism of O-glycosylation. We find that loss of pgant5 causes irregular secretory granule morphology, altered mucin packaging, and disruption of secretion. Alternative splicing of pgant5 yields two isoforms, pgant5A and pgant5B, with higher expression of pgant5A in salivary glands. Rescue with pgant5A, but not pgant5B, partially restores secretory granule morphology and salivary gland function. A single residue difference within the substrate binding pockets of PGANT5A and PGANT5B increases the in vitro activity and specificity of PGANT5A towards salivary gland mucins relative to PGANT5B, corroborating the in vivo expression and role of pgant5A in salivary glands. Our data additionally provide new insights into a catalytic domain motif that regulates GalNAc-T activity. Overall, our studies illustrate how alternative splicing of an O-glycosyltransferase that results in modest amino acid changes can influence substrate specificity to regulate tissue-specific biological functions.

biochemistry↗

bioRxiv2025

Theta-mediated conceptual reinstatement in vmPFC precedes perceptual reinstatement in ventral visual cortex during memory recall

When recalling past episodes, different features of an experience, such as conceptual meaning and perceptual detail, are reconstructed and reinstated across distributed cortical regions. However, current models of human memory remain unclear how these feature-specific reinstatements unfold over time, and whether they interact hierarchically during memory retrieval. Using magnetoencephalography (MEG) and time-resolved encoding-retrieval cross-phase classification analysis, we compared the time course of conceptual and perceptual reinstatement during cued visual recall. Conceptual information decoding in the ventromedial prefrontal cortex (vmPFC) preceded perceptual information decoding in the ventral visual cortex (VVC), and the two were expressed in distinct frequency bands: theta (4-8 Hz) for conceptual and gamma (30-40 Hz) for perceptual information. Cross-correlation and spectral Granger causality analyses revealed that during reinstatement conceptual information in vmPFC predicted and directed perceptual information in VVC, with theta-band information flow predominantly from vmPFC to VVC. In addition, bottom-up connectivity from VVC to vmPFC was expressed in the alpha band. These findings suggest that memory retrieval proceeds from abstract conceptual reconstruction to the reinstatement of perceptual details, mediated by theta oscillatory communication between prefrontal and sensory areas. We propose a hierarchical interactive model in which vmPFC initiates conceptual activation through theta-band top-down signals, while VVC provides alpha-band feedback for evaluating reconstructed perceptual details.

neuroscience↗

bioRxiv2025

Two Small Proteins Enable N2O Reduction in Acidic Environments

Denitrifying bacteria are major contributors to nitrous oxide (N2O) emissions, which rise with global soil acidification, likely because low pH impairs maturation of the periplasmic enzyme N2O reductase (NosZ) in most bacteria. While some bacteria can reduce N2O under acidic conditions, the underlying mechanism remain unclear. Here, we isolated bacteria from acidic soils and identified strains that efficiently reduce N2O at low pH. They carry two novel proteins, NosP and NosQ, along with NosZ clade I. Functional analyses of deletion mutants demonstrated that NosP and NosQ are essential for the maturation of NosZ at low pH, but are not required at circumneutral pH. Cryo-EM analysis indicates that NosPQ forms a protective complex with NosZ, safeguarding its maturation under acidic conditions. Metagenomic analysis shows that nosPQ genes are widely distributed across diverse bacterial taxa and soil types. Our findings reveal a key molecular adaptation mechanism for N2O reduction in acidic environments and provide valuable insights for developing efficient N2O mitigation strategies.

biochemistry↗

bioRxiv2025

TUPAD: A Tn5-Mediated, Pre-amplification/Adapter-Dimer-Free Platform for Ultra-Uniform, One-Step PGT Profiling with High Stability and Broad Compatibility

High-throughput sequencing stands as a cornerstone of clinical molecular diagnostics, particularly in preimplantation genetic testing (PGT)--a key application where the selection of chromosomally normal embryos is critical to enhancing in vitro fertilization (IVF) success rates. A fundamental challenge inherent to PGT, however, lies in the ultra-low gDNA input (typically <100 pg) from embryo biopsies. To mitigate this limitation, traditional workflows rely on whole genome amplification (WGA) technologies, such as MDA, PicoPlex, and MALBAC. Yet these WGA-based protocols inherently entail pre-amplification of gDNA prior to standard library construction---a step that introduces significant coverage bias and compromised uniformity. These are intrinsic flaws irreparable by downstream library preparation or bioinformatics analysis, ultimately leading to clinical uncertainty in the interpretation of copy number variations (CNVs). In this study, we developed TUPAD (Tn5-Mediated, Uniform Pre-amplification-free and Adapter-Dimer-Free Library preparation), a transformative technology that bypasses the limitations of traditional WGA. By engineering a novel adapter-dimer-free transposon system, TUPAD enables high-efficiency library construction from as little as 1 picogram (pg) of gDNA. This breakthrough allows for a pre-amplification-free workflow, effectively eliminating the primary source of amplification bias. Compared to existing WGA standards, these advantages are fully retained in embryo biopsy and positive sample testing, TUPAD consistently generates highly uniform library data, significantly enhancing the detection limit and accuracy of CNVs and other genomic variants. Beyond these performance advantages, TUPAD also cuts wet-lab processing time by 50%. Practical validation confirms TUPAD results are stable, reliable, with proven practical utility in PGT testing.

molecular biology↗

bioRxiv2025

Echoes as Signal, Not Noise: Reverberation Sharpens Sensitivity to Temporal Structure

Sensitivity to temporal structure is fundamental to hearing, yet how natural reverberation shapes this sensitivity is unclear. Here we identify echo-facilitated temporal sensitivity (EFTS), a principle whereby the auditory system exploits echoes to selectively enhance responses to rapid temporal changes. Using transitional click trains with subtle inter-click-interval (ICI) shifts, we first show that increasing echo strength systematically enlarges the sound-level step at ICI transitions in both recorded and simulated stimuli, sharpening the physical boundary between segments of distinct temporal structure. In humans, psychophysical detection of ICI changes improves monotonically with echo level in both simulated and real free-field environments, while a late EEG change response scales with echo strength as onset responses remain comparatively stable. In awake rats, electrocorticography over auditory cortex reveals parallel echo-dependent enhancement of transition-evoked activity. Single units exhibit echo-level-dependent amplification of change-related firing and improved neurometric discriminability. Laminar local field potential and current source density analyses further show that echo-dependent divergence emerges first in granular and infragranular layers before propagating to supragranular cortex, consistent with thalamocortical drive followed by intracortical amplification. Together, these findings establish EFTS as a cross-species mechanism that reframes echoes as structured signals the brain uses to sharpen temporal integration in everyday listening.

neuroscience↗

bioRxiv2025

Mapping Core Components of Membrane-less Organelles in Living Cells by Phase-APEX2 Proximity Labeling

Membrane-less organelles (MLOs), formed via liquid-liquid phase separation, are crucial for organization of biomacromolecules and cellular processes, yet their dynamic nature challenges compositional analysis. Conventional proximity labeling methods lack the specificity to overcome high background and systematic biases inherent in studying these condensates. Here we introduce Phase-APEX2, a three-input AND-gate strategy that dramatically enhances labeling specificity. By integrating a split-APEX2 system with a light-controlled H2O2 generator, we developed Phase-APEX2 platforms for two application contexts: Phase-APEX2-MS for proteomics and Phase-APEX2-seq for genomics. Our analyses revealed not only the core interactomes of the nucleolus and stress granules with high accuracy but also uncovered distinct "molecular grammars" governing their assembly and a surprising link between nucleolar dynamics and the transcriptional regulation of mitosis-related genes. This highly specific and adaptable platform opens new avenues for exploring the composition and function of diverse biomolecular condensates.

cell biology↗