Biology subjects

Zhu, W.

Publications and source records attributed to Zhu, W..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Human TBC1 domain-containing kinase is a class I multidomain pseudokinase

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

biochemistry↗

bioRxiv2026

Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS

Current methods for the imaging of glycosylated RNAs (glycoRNAs) focus on limited types with identified RNA sequences, making the distribution of global glycoRNAs at single-cell and tissue levels largely unexplored. To address this gap, we developed a co-metabolic proximity labeling assay (COMPASS) for sequence-independent imaging of glycoRNAs. COMPASS employs metabolic chemical reporters (MCRs) to label glycans and RNAs, followed by click-chemistry-based dye conjugation to generate Forster resonance energy transfer (FRET) signals. The MCR-enabled RNA labeling allows for sequence-independent imaging, and FRET ensures accurate localization of glycoRNA distributions. COMPASS facilitates glycoRNA imaging across multiple cell lines, cerebral organoids, and mouse tissues. We observed the downregulation of glycoRNAs in metastatic lung nodules and upregulation in original regions of brain development in cerebral organoids, suggesting their potential association with carcinogenesis and neurogenesis. COMPASS illuminates previously invisible glycoRNAs and offers a comprehensive profile of glycoRNA distribution for studying their functions.

bioengineering↗

bioRxiv2026

A novel C57BL/6 mouse model for the study of severe Citrobacter rodentium infection

The study of human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) has been limited by the inability of these pathogens to effectively colonize murine models without prior antibiotic treatment. Because it mimics key features of human EPEC and EHEC infection, Citrobacter rodentium, a natural mouse pathogen that colonizes the lower intestine, has become the primary model for investigating these organisms. C57BL/6 mice are most commonly used for C. rodentium research, however, unless they carry specific genetic mutations, they typically develop only mild disease and clear the infection within weeks. As a result, models of severe disease in genetically unmodified hosts are lacking. Here, we describe the development of a non-genetically modified C57BL/6 mouse line with an undisturbed intestinal microbiota, highly susceptible to severe C. rodentium infection. Early infection in these mice was marked by significantly elevated cecal bacterial burdens and tissue pathology. Immune profiling revealed broad reductions in multiple lymphoid subsets, indicating impaired early mucosal activation. Although overall cytokine expression patterns were similar between groups, ceca of susceptible mice exhibited elevated baseline and early post-infection IL-18, as well as increased G-CSF at day 1. Microbiota analyses showed broadly comparable communities with wildtype controls, with some altered groups, such as Lachnospiraceae, Prevotellaceae, Desulfovibrionaceae, and Erysipelotrichaceae. Together, these findings characterize a robust C57BL/6 model that reproducibly develops severe C. rodentium-induced disease. This phenotype is driven by microbiota-associated alterations and impaired early cecal immunity, providing a valuable system for studying host-microbiota interactions in enteric infections.

microbiology↗

bioRxiv2026

Decoding epitope immunodominance in HIV Env using cryoEM and machine learning

Viral surface glycoproteins, such as the HIV envelope protein (Env), present numerous antibody (Ab) epitopes, yet immune responses consistently focus on only a subset, a phenomenon known as immunodominance. Although structural studies have provided insights into Env antigenicity, our understanding of the molecular features that govern efficient Ab engagement remains incomplete, thereby limiting the predictive and rational design of vaccines. Here, we characterized the structural determinants of epitope immunodominance in HIV Env by integrating high-resolution cryoEM-based polyclonal epitope mapping (cryoEMPEM) across different clades with quantitative analyses of epitope topology, accessibility, and physicochemical properties. More than 70 new structures were resolved to assemble a library of >100 Env-antibody complexes. These data informed the development of a surface-centric, machine-learning model to predict relative Antigen Surface Immunodominance (ASI model). Comparison of ASI-predicted epitope sites with the specificities of Env-induced antibodies showed that the model accurately identifies immunodominant regions and highlights the structural features driving immune bias. Notably, immunogens redesigned based on model predictions successfully redirected Ab responses toward a normally subdominant epitope, demonstrating the potential of strategies coupling targeted assembly of focused structural libraries with machine learning to uncover complex molecular patterns and enable design of more effective vaccine antigens.

immunology↗

bioRxiv2026

Single-phage profiling illuminates viral individuality during cell fate determination

The choice between cell death (lysis) and viral dormancy (lysogeny) following bacteriophage infection serves as a founding paradigm for the emergence of cellular heterogeneity in a genetically uniform population. The determination of host fate arises through the stochastic transcription from multiple viral genomes present within each cell, but this activity remains hidden from empirical interrogation, which typically stops at the whole-cell level. Here we use parallel sequential fluorescence in situ hybridization (par-seqFISH), followed by spatial clustering of phage-encoded transcripts within each cell, to profile the transcriptional activity of individual phages during synchronized infection of Escherichia coli (E. coli) by bacteriophage lambda. At the whole-cell level, transcription kinetics capture the developmental choice between lysis and lysogeny, and further demonstrate that viral replication is required for the emergence of diverging fate decisions. Zooming in to the single-phage level illuminates an individuality of viral activity during infection. We find that, while cells pursuing lysogeny display consensus activity of all in-habiting phages, lytic cells may contain phages that exhibit lysogenic activity. These findings support an earlier suggestion that consensus among coinfecting phages is required for cell dormancy. More broadly, our results highlight the need to identify how whole-cell behavior emerges from the activity of physically distinct copies of the same genetic circuit.

systems biology↗

bioRxiv2026

Reaction-enabled, highly sensitive super-resolution imaging

Super-resolution microscopy breaking the optical diffraction limit has led to success in illuminating the complexity of biology with fluorescence--light-excited luminescence1,2. Here, we propose another approach for super-resolution imaging with electrochemiluminescence (ECL)--reaction-excited luminescence3,4. While featuring high sensitivity in molecular analytics owing to its laser-free excitation, the current reaction-driven imaging suffers from limited resolution due to low photon budget4. Efficient ECL imaging of intracellular organelles remains elusive. To meet this challenge, we develop RIED (Reaction-based super-resolution Imaging via Entropy-weighted correlation combined with Deconvolution), which builds upon two pivotal advancements: (i) unprecedented ECL intracellular organelles imaging through a 1,000-fold enhancement in ECL emission, and (ii) the super-resolution ECL imaging by the spatiotemporal acquisition and efficient utilization of highly dynamic ECL profiles. RIED allows for the first three-dimensional super-resolution ECL imaging with a spatial resolution of {bsim}97 nm in the lateral dimension and {bsim}235 nm in the axial dimension. This allows to reveal outer mitochondrial membrane topology and distinct microtubule organizations in different mitosis phases, offering a chemistry-based imaging capability for high-throughput imaging and highly sensitive interfacial imaging, which provides a new dimension into biology.

biochemistry↗

bioRxiv2026

A motif-vocabulary model of CAR T-cell intracellular domains identifies determinants of immunophenotype differentiation

Chimeric antigen receptor (CAR) T-cell efficacy depends critically on the costimulatory domain, which shapes downstream signaling and the immunophenotype of manufactured products. Despite mechanistic evidence that immune receptors function as motif-based signaling scaffolds, CAR engineering has focused on a narrow set of costimulatory domains--principally CD28 and 4-1BB--leaving much of the available signaling design space unexplored. Here, we screened 1,243 naturally occurring intracellular domains as costimulatory modules in an anti-CD20 CAR backbone in primary human CD8+ T cells and quantified construct enrichment across memory-differentiation and PD-1-defined immunophenotypic compartments. Using Eukaryotic Linear Motif (ELM) annotations, we analyzed motif-phenotype associations via complementary statistical approaches: Mann-Whitney screening and negative binomial regression identified ELM features associated with differential construct representation, while Dirichlet-Multinomial modeling--which properly accounts for the compositional structure of FACS-partitioned data--revealed that individual ELMs do not significantly alter phenotype distributions. This discrepancy indicates that single motifs primarily affect proliferation or survival rather than differentiation fate. In contrast, construct-level analysis using a leave-one-out compositional test identified specific costimulatory domains with significant phenotype-shifting effects, demonstrating that particular combinations of ELMs--rather than individual motifs--determine immunophenotype. These results suggest that CAR T-cell differentiation state is governed by the integrated output of multiple signaling motifs and provide a combinatorial framework for rational costimulatory domain engineering.

bioengineering↗

bioRxiv2026

Conformational Remodeling Underlies Activity Loss in Disease-Linked Asparagine Synthetase Variant

Asparagine synthetase deficiency (ASNSD) is a devastating congenital disorder characterized by profound neurological impairment and early childhood mortality. It is associated with pathogenic mutations in the asparagine synthetase (ASNS) gene. Despite the critical role of ASNS in the amino acid cycle, the molecular basis by which ASNSD-linked missense mutations impair enzyme function remains poorly understood. Here, we present a comprehensive characterization of a recurrent ASNSD-linked variant, R48Q. Steady-state kinetic assays reveal severe reductions in L-glutamine-dependent catalysis and disrupted product stoichiometry, implicating impaired interdomain communication. Cryogenic electron microscopy (cryo-EM) and 3D variable analysis of the EM map uncovers altered loop conformations at the N-terminal active site and subtle conformational changes at the C-terminal domain. Consistent with the structural data, molecular dynamics simulations support that the local disruption propagates across the protein, thereby decoupling coordinated domain motions essential for catalysis. Additionally, we demonstrate that the flanking arginine and the affected loop are evolutionarily conserved across Class II glutamine amidotransferases, highlighting their shared mechanistic importance. These findings provide the molecular basis of an ASNSD variant and establish a framework for understanding how point mutations disrupt complex enzyme dynamics, with broad implications for precision medicine. SignificanceUnderstanding how mutations affect multidomain enzymes is crucial for elucidating the molecular mechanisms underlying genetic disorders. Here, we examine the molecular consequences of the R48Q variant in human asparagine synthetase (ASNS), the sole enzyme responsible for de novo L-asparagine synthesis; mutations of this enzyme lead to a fatal neurometabolic disorder, asparagine synthetase deficiency (ASNSD). By combining biochemical, cryogenic electron microscopy, and molecular dynamics simulation, we show that a single N-terminal amino acid substitution disrupts both local and global coordination, impairing enzyme activity. Our work provides the first mechanistic blueprint of an ASNSD-linked variant. These findings not only deepen our understanding of ASNS but also offer a generalized framework for studying the dynamic regulation of multidomain enzymes in disease.

biochemistry↗

bioRxiv2026

Mismatch tolerance of a gRNA for CRISPR-based gene activation confers broad activity critical for cell reprogramming

CRISPR activation and interference systems (CRISPRa/i) are widely used for programmable transcriptional control. Although these technologies are capable of highly specific single-gene activity, some applications of transcriptional network reprogramming require broad, genome-wide effects. Here, we identify a CRISPRa gRNA that robustly reprograms astrocyte transcriptional state. Unexpectedly, this activity arises from extensive off-target binding that induces expression changes in thousands of genes, unlike neighboring gRNAs targeting the same intended on-target site. We leverage this promiscuous gRNA to dissect determinants of gRNA-driven off-target dCas9 binding in the context of transcriptional reprogramming. Using ChIP-seq, high-throughput protein-binding microarrays, and gRNA-variant library screening in cells, we demonstrate that PAM-proximal bases are primary determinants of genomic binding, mismatch tolerance is both gRNA- and base-specific, and targeted mutations within the PAM-proximal region can tune gRNA specificity. We further demonstrate that CRISPRa-driven phenotypes can reflect combined contributions from widespread off-target activity and dose-dependent on-target effects. These findings highlight the potentially widespread impacts of CRISPRa off-target activity, underscore the need to account for cryptic effects when selecting and evaluating gRNAs for programming cell phenotypes, and demonstrate that multi-site binding by CRISPRa systems can be exploited as a feature for network-level perturbations in cell reprogramming. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/703129v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b697b0org.highwire.dtl.DTLVardef@1a0b390org.highwire.dtl.DTLVardef@16ce710org.highwire.dtl.DTLVardef@b5d87a_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Beyond Lipschitz: Ranking Binding Affinity in Hyperbolic Space

Despite the importance of protein-ligand affinity ranking in drug discovery, existing deep learning models struggle to distinguish hard inactives that are structurally similar to active compounds but biologically inactive. We theoretically show this failure stems from the Lipschitz continuity constraint in Euclidean space, which makes neural networks locally insensitive to subtle yet critical structural perturbations. To overcome this, we propose AlphaRank, which demonstrates that scoring affinity as the negative hyperbolic geodesic distance allows subtle tangential variations to bypass Lipschitz constraints, intuitively corresponding to pivotal binding mode factors such as conformational fit. Meanwhile, AlphaRank employs joint optimization to simultaneously ranking affinity scores among actives in a proximity-aware manner and separate actives from decoy inactives through a geometric cone constraint. Experiments demonstrate that AlphaRank outperforms state-of-the-art models like Boltz-2 in both affinity ranking and active-inactive discrimination, while providing a interpretable representation space.

bioinformatics↗

bioRxiv2026

Moraxella catarrhalis HemW is a Heme-binding Radical SAM Enzyme

Moraxella catarrhalis is an emerging human respiratory pathogen responsible for a significant proportion of childhood otitis media and exacerbations of chronic obstructive pulmonary disease. Recent transposon sequencing analysis identified yggW (renamed as hemW), an uncharacterized gene, as essential for M. catarrhalis growth under iron-limiting conditions, mimicking host-imposed nutritional immunity. HemW is annotated as a putative radical S-adenosylmethionine (SAM) enzyme and belongs to the HemN-like subfamily, but its biochemical properties remain unclear. Here, we report on the first experimental characterizations of McHemW. Our bioinformatic analysis confirmed its evolutionary relationship with the putative heme-binding radical SAM enzyme EcHemW in Escherichia coli. Using biochemical and spectroscopic approaches, we demonstrate that McHemW contains a catalytically active [4Fe-4S] cluster and binds heme in vitro. These findings support a functional role for McHemW as a putative heme chaperone, highlighting it as a potential target for disrupting iron metabolism in this clinically important pathogen.

biochemistry↗

bioRxiv2025

Development of a point-of-care field diagnostic test for DFT1 and DFT2

The Tasmanian devil (Sarcophilus harrisii) population has undergone a major decline in the wild due to the epidemics of two transmissible cancers known as devil facial tumours 1 (DFT1) and devil facial tumour 2 (DFT2). Understanding the distribution and prevalence of DFT1 and DFT2 is challenging as they can only be detected in field conditions when tumours are large enough to be visually observed. This impedes our ability to understand how the devil population is evolving in response to the tumours. PCR-based diagnostics are available, but the technique is not field-applicable. To overcome these hurdles, we developed an ultrasensitive and field-deployable rapid diagnostic tool. We used a technique called Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK), which combines an isothermal nucleic acid amplification step with CRISPR-based recognition and activation of reporter probes to indicate target sequence detection. This system requires only portable laboratory equipment, making it more amenable to field conditions. The results showed that our test can detect DFT1 and DFT2 DNA within the attomolar range. We then confirmed that the test can detect DFT1 in the field using non-invasive swab samples. The SHERLOCK test can be used for more active monitoring and management approaches that are not currently possible.

molecular biology↗

bioRxiv2025

α-Parvin regulation of cell re-arrangement is critical for ureteric bud branching morphogenesis

All branched tubular structures, including the kidney collecting system, are formed by branching morphogenesis, a process that includes tip branching and trunk narrowing. Tight control of cell movement and rearrangements is a prerequisite for branching morphogenesis. The role of integrin-associated adhesion proteins in coordinating actin dynamics and cell rearrangements during branching morphogenesis is poorly understood. Here we used 3D live imaging of mouse ureteric bud branching to show that -parvin, a component of the integrin binding ILK-PINCH-Parvin (IPP) complex, regulates tip branching and tubule thinning by inhibiting excessive cell adhesion and actin polymerization. Mechanistically, -parvin promotes actin turnover by inhibiting activation of the small GTPases RhoA and Cdc42, which in turn enhances the severing function of the actin regulatory protein, cofilin. These results underscore the importance of adhesion protein-regulated actin dynamics in the critical process of cell rearrangement, which is required for branching morphogenesis. TeaserScaffold protein -parvin limits cell adhesion and actin polymerization, enabling cell rearrangements that drive kidney branching

developmental biology↗

bioRxiv2025

Hemin Inhibits the Activation of STING in Macrophages by Inducing HO-1, Promoting Endometriosis Development

IntroductionEndometriosis (EMS) is an estrogen-dependent inflammatory disorder characterized by immune dysregulation. This condition profoundly affects the quality of life and reproductive health of nearly 200 million women of reproductive age worldwide and poses significant clinical challenges due to lack of diagnosis tools and effective treatment. Peritoneal macrophages play a central role in promoting the initiation and progression of EMS. Bioinformatics studies have indicated that the STimulator of INterferon Gene (STING) was downregulated in the macrophages in EMS, and this influenced their polarization and functional. However, the specific contribution of STING pathway alterations to EMS pathogenesis remains unclear. MethodsWe integrated scRNA-seq data from EMS patients and healthy controls-including lesions, eutopic endometrium, non-lesional tissue, and peritoneal fluid from EMS patients and healthy controls(n = 19,291 total samples)-to analyze macrophage STING expression and function. Mouse EMS model was generated by injecting uterus fragments into WT or STING-/- mice. Peritoneal fluid was collected and macrophage proportions and subtypes were assessed via flow cytometry. Macrophages were depleted in STING-/- mice prior to EMS induction to evaluate their role in disease progression. In vitro, hemin was used to treat macrophages to investigate how heme oxygenase-1 (HO-1) modulates the STING pathway, assessed through immunoblotting, co-immunoprecipitation, ELISA, and multiplex immunofluorescence. ResultsBioinformatic analysis revealed that macrophages within the EMS lesions are primarily derived from peritoneal macrophages and exhibit decreased STING expression, which correlated negatively with HO-1. STING-/- mice developed more numerous and larger EMS lesions, accompanied by a decreased proportion of large peritoneal macrophages and an increase in small peritoneal macrophages. A marked elevation in M2-type macrophages within the lesions was detected in STING-/- mice. In vitro, hemin-induced HO-1 suppressed STING pathway activation in macrophages. Mechanistically, HO-1 inhibited the translocation of STING from the endoplasmic reticulum to the Golgi, thereby suppressing STING signaling and facilitating EMS. ConclusionMacrophages are essential for lesion formation in STING-/- mice. In vitro, hemin promotes EMS progression via HO-1 - mediated suppression of the STING pathway. Our findings identify the heme-HO-1-STING axis as a key immunomodulatory pathway in EMS and suggest its potential as a therapeutic target. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/683839v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@6901beorg.highwire.dtl.DTLVardef@1f18d44org.highwire.dtl.DTLVardef@1716f6forg.highwire.dtl.DTLVardef@4ae22d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract (Created in BioRender.com) Retrograde menstruation leads to the accumulation of heme in the peritoneal cavity, which upregulates HO-1 expression in peritoneal macrophages. HO-1 inhibits the translocation of STING from the endoplasmic reticulum to the Golgi apparatus in macrophages, thereby suppressing the STING pathway and promoting the development and progression of EMS. The progression of EMS, in turn, reinforces this mechanism, creating a self-sustaining vicious cycle. C_FIG

immunology↗

bioRxiv2025

Normative models of individualized functional brain networks reveal language network expansion in autism

Autism spectrum disorder is a highly heterogeneous neurodevelopmental disorder, hindering mechanistic insights and the identification of biomarkers for clinical diagnosis. Recently, precision functional mapping has been developed to identify abnormalities in brain network topologies associated with various psychiatric disorders, yet its application in autism remains limited. Here, we utilized precision functional mapping and a large, multisite neuroimaging dataset (N = 1,182) to construct individualized functional networks in individuals with autism. We developed normative models using network surface area from healthy controls (n = 628) to characterize typical brain network organization across age, allowing for the quantification of individual-specific deviations in individuals with autism (n = 554). We found widespread and heterogeneous deviations from the normative model, with the language network emerging as the most significantly altered region, thereby emerging as an epicenter of functional disruption in autism. Individuals with autism were clustered into three subtypes involving distinct functional network topologies, associated with behavioral profiles marked by impairments in perception, language processing, or socio-emotional functioning. We further linked these atypical brain features to cortical gene expression patterns, revealing enriched pathways related to neurodevelopment, language, and signaling processes. Together, these findings reveal autism-specific deviations in individualized functional brain networks, offering potential clinical relevance for understanding and stratifying autism.

neuroscience↗

bioRxiv2025

Order-Selective Cells Tile Temporal Space and Predict Order Memory in Humans

Remembering the temporal order of events is critical for episodic memory, allowing us to link individual events into sequences. While the medial temporal lobe and prefrontal cortex are essential for this process, the underlying neural mechanisms remain poorly understood. Here we characterized the representation of order information at the level of single neurons and field potentials recorded from human neurosurgical patients watching naturalistic videos of everyday events and later recalling the order and content of the events depicted. We found order-selective cells (OSCs) in the human hippocampus, amygdala, and orbitofrontal cortex that responded selectively to specific event orders, independent of event content or absolute time. OSCs exhibited transient theta phase precession following their preferred order during both memory encoding and retrieval, the strength of which predicted participants order memory accuracy. During retrieval, OSC spike timing relative to theta varied with the relative position of their preferred event within the recalled event sequence, enabling selective retrieval of relevant events. These findings reveal a neural substrate for representing, encoding into and retrieving from memory absolute and relative ordinal relationships between discrete events. OSCs tile temporal space into discrete ordinal positions, thereby weaving episodic experiences into coherent temporal narratives.

neuroscience↗

bioRxiv2025

Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response

Targeting asparagine metabolism is a promising strategy for treating asparaginase-resistant acute lymphoblastic leukemia (ALL), sarcoma, and potentially other solid tumors. Here, we characterize the molecular mechanism by which a cell-penetrable small molecule, ASX-173, inhibits human asparagine synthetase (ASNS), the enzyme that catalyzes intracellular asparagine biosynthesis. ASX-173 reduces cellular asparagine levels, induces the integrated stress response (ISR), and reduces cell growth in HEK-293A cells. A cryo-EM structure reveals that ASX-173 engages a unique, hydrophobic pocket formed by AMP, Mg2+, and pyrophosphate in the C-terminal synthetase domain of ASNS, thereby enabling multivalent, high-affinity binding. Based on in vitro kinetic and thermal shift assays, we find that ASX-173 binds to the ASNS/Mg2+/ATP complex and is therefore a rare example of an uncompetitive enzyme inhibitor with potential therapeutic use. These findings provide a structural and mechanistic basis for targeting ASNS with small molecules, which have application in treating cancer and other human diseases.

molecular biology↗

bioRxiv2025

Observing biological spatio-angular structures and dynamics with statistical image reconstruction and polarized fluorescence microscopy

Understanding molecular orientation and density distributions is essential for exploring biological structure and function. Polarized fluorescence microscopy (PFM) provides insights into molecular architecture but struggles to resolve three-dimensional (3D) molecular orientation distributions, particularly in densely labeled or structurally complex specimens. To address this, we introduce the efficient generalized Richardson-Lucy (eGRL) algorithm, a robust framework for reconstructing 3D molecular density and orientation (spatio-angular) distributions from PFM data. By modeling the imaging process in spatio-angular hyperspace, we propose a maximum-likelihood solution enhanced by dimensionality reduction and angular domain transformation to overcome computational challenges. eGRL improves accuracy and efficiency across different PFM implementations, enabling use on standard platforms. We utilize our methods to resolve biological spatio-angular structures and dynamics otherwise impossible to resolve, including the tangential alignment of actin filaments in U2OS cells, nanowire-guided cytoskeletal organization in NIH3T3 cells, rotational actin patterns in live HeLa protrusions, and membrane tension-induced anisotropy in live macrophages.

bioengineering↗