Biology subjects

Fan, M.

Publications and source records attributed to Fan, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Hydrogel dimensionality directs mechanics-dependent nascent matrix deposition by tendon fibroblasts

Tendon fibroblasts continuously synthesize and remodel extracellular matrix (ECM) in response to their surrounding mechanical environment in vivo and in vitro, yet how dimensionality regulates the deposition of newly synthesized matrix remains poorly understood. Here, we investigated nascent matrix deposition by primary mouse tail tendon fibroblasts cultured on flat two-dimensional (2D) or encapsulated within three-dimensional (3D) norbornene-modified hyaluronic acid hydrogels with tunable mechanical properties. Newly synthesized matrix was visualized using metabolic labeling with azido-galactosamine (GalNAz), which labels nascent glycoproteins deposited during culture. In 2D, increasing hydrogel elastic modulus from 10 to 25 kPa enhanced nascent matrix deposition. This response was accompanied by increased cell spreading and YAP nuclear localization, indicating increased cellular mechanosensing. In contrast, tendon fibroblasts encapsulated within enzymatically degradable 3D hydrogels showed progressive matrix deposition over time but deposited less nascent matrix in stiffer (~50 kPa) hydrogels compared with softer 5 and 10 kPa hydrogels. YAP nuclear localization was increased in softer hydrogels compared with 50 kPa hydrogels. Bulk RNA sequencing showed increased expression of tendon matrisome-associated genes in softer 3D hydrogels, including increased tenascin C and collagen type I expression. These findings demonstrate that hydrogel dimensionality determines the relationship between matrix mechanics and nascent matrix deposition. This work establishes a framework for studying how engineered microenvironments regulate the matrix-producing behavior of tendon fibroblasts and provides insight into the design of biomaterials for investigating tendon biology and matrix remodeling.

bioengineering↗

bioRxiv2026

SIGMA: SIgnaling via Golgi Morphology Analysis for pathway-state classification

Organelle morphology is increasingly recognized as a computationally accessible readout of molecular state, yet whether subcellular architecture can identify which signaling pathway is active, not merely whether a cell is perturbed, remains untested. Here we introduce SIGMA (SIgnaling via Golgi Morphology Analysis), a framework that reframes pathway identification as a morphological classification problem centered on the Golgi apparatus. SIGMA integrates imaging flow cytometry for population-scale Golgi profiling, teacher-student knowledge distillation to transfer fine morphological cues from limited high-resolution images to an efficient lower-resolution classifier, and mechanistic generalization testing with chemically distinct activators. Applied to MRC-5 fibroblasts across six conditions spanning four signaling cascades plus controls, SIGMA achieved 90.02% cell-level accuracy and correctly assigned five of seven structurally unrelated external compounds to their expected pathway class. These results establish Golgi morphology as a multiplexed, machine-readable fingerprint of signaling state.

molecular biology↗

bioRxiv2026

Scalable spatial DNA sequencing from archival tissue maps copy number subclones

Spatially resolved DNA sequencing holds promise due to its potential utility in understanding cancer intra-tumour heterogeneity and tumour evolution in relation to tissue architecture. However, it has so far been used to a limited extent due to technical challenges and high cost of existing methods. Hence we aimed to develop a high throughput spatial genomic assay to obtain copy number alteration (CNA) information at user-defined spatial resolution. We derived CNA profiles from ultra-low coverage whole genome sequencing at sub-millimetre resolution from archival samples using a novel method called Adaptive Resolution Multiscale Spatial DNA sequencing (ARMS DNAseq). We used it to profile CNAs from more than 766 regions (tiles) from 3 patients, covering a total area of over 300 mm2, with 1.2-2.6 million mapped reads per tile and tile sizes of 0.1-0.99mm2. Using ARMS DNAseq, we delineate tumour evolution in a spatial context, and identify more tumour subclones that were obscured or incompletely represented in bulk multi-region whole genome sequencing. Next, we show associations between tumour subclones and morphology, and prediction of subclone identity from deep learning-derived image representations. Finally, we demonstrate multi-omic integration by alignment with spatial transcriptomic data, showing subclone-specific immune cell co-occurrence as well as transcriptional programmes cutting across subclone boundaries. ARMS DNAseq converts low-throughput, region-by-region profiling into a scalable and adaptable workflow for direct spatial copy number profiling from archival tissue sections.

genomics↗

bioRxiv2026

Decorin-mediated Extracellular Matrix Stabilization Maintains Cartilage Integrity During Aging

Aging is a primary risk factor for osteoarthritis (OA), yet the mechanisms that preserve extracellular matrix integrity in long-lived connective tissues remain poorly defined. The function of articular cartilage depends on maintaining extracellular matrix integrity over a lifetime of mechanical use. Here we identify failure of matrix stabilization as an initiating mechanism of age-associated OA. Cartilage-specific deletion of decorin in mature mice disrupted the superficial collagen II fibrillar network, reduced aggrecan retention, and impaired poroelastic fluid pressurization by 9 months of age, preceding substantial transcriptional changes in resident chondrocytes. With advancing age, these matrix defects culminated in cartilage erosion, fibrotic remodeling, and spontaneous OA by 18 months. Mechanistically, decorin attenuated force-induced collagen II fibril realignment and reinforced the superficial fibrillar network, preserving matrix architecture under sustained physiological loading. These findings establish decorin-mediated extracellular matrix stabilization as a critical determinant of cartilage longevity and support a matrix-first model of age-associated degeneration.

biophysics↗

bioRxiv2026

Catch-bond engineering surpasses high-affinity maturation for T cell receptor therapies against solid tumors

The sensitivity of T cell receptors (TCRs) has traditionally been attributed to their affinity, a principle that has long underpinned both T cell biology and TCR engineering. Currently, high-affinity maturation remains the predominant strategy employed to enhance TCR sensitivity; however, this approach has been associated with severe off-target toxicities in clinical settings. In this study, using the only FDA-approved TCR-T therapy as a clinical reference, we demonstrate that force-induced catch bonds, rather than static affinity, primarily determine TCR sensitivity and facilitate the development of more effective TCR engineering strategies. Among polar and charged amino acids, histidine was determined to be the most effective residue for pinpointing engineering hotspots. TCRs engineered to form catch bonds exhibited superior performance compared to the FDA-approved TCR subjected to high-affinity maturation, improving the efficacy of TCR-T cell therapies against solid tumors without eliciting off-target toxicity or alloreactivity. Mechanistically, a correlation was observed between TCR sensitivity and the strength of catch bonds, whereas no such relationship was found with affinity. The duration of T cell-tumor cell interactions, immunological synapse formation, and the intensity of subsequent signaling are governed by catch bonds rather than by affinity. Structural and computational investigations have demonstrated that force-induced reconfiguration of the ligand-receptor interface, along with the formation of a novel hydrogen bond network, augments the specificity of interactions between the TCR and antigenic peptides. Furthermore, bispecific T cell engagers utilizing the TCR scaffold also formed catch bonds with tumor antigens, indicating intrinsic mechanosensory properties of the extracellular domains of TCRs and suggesting a novel therapeutic modality based on catch-bond-engineered TCRs.

immunology↗

bioRxiv2026

Internalized Components of Membrane Attack Complexes Disrupt Proteostasis and Acquire Alarmin-Like Properties

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

biochemistry↗

bioRxiv2026

Magnetically guided macrophage immunobots coordinate iron-metabolic cascades and immunogenic ferroptosis for tumor immunotherapy

Adoptive cell therapy (ACT) remains challenging in solid tumors, where poor tumor infiltration, metabolic heterogeneity, and an immunosuppressive tumor microenvironment (TME) constrain therapeutic efficacy. Here, we developed a magnetically actuated macrophage-based immune microrobot (immunobot) for active solid tumor immunotherapy. Immunobots were constructed by loading bone marrow-derived macrophages (BMDMs) with lipopolysaccharide-modified Janus L1-FePt magnetic microrollers (LMRs), enabling hard-magnetic actuation. Optimized LMR loading supported robust propulsion, retention under flow, and enhanced barrier penetration. LMRs further promoted M1-like polarization through LPS-driven inflammatory activation and FePt-derived labile iron-amplified oxidative stress, with altered iron homeostasis, increased reactive oxygen species (ROS), and enhanced NF-{kappa}B signaling. Immunobots also induced ferroptosis-associated immunogenic cell death in tumor cells. In vivo, magnetically guided immunobots suppressed tumor growth, reprogrammed tumor-associated macrophages (TAMs), promoted dendritic cell maturation, and enhanced CD8 T cell activation. This work establishes a microrobotic immunotherapy platform for active magnetic transport, iron-metabolic regulation, and immune remodeling in solid tumors.

bioengineering↗

bioRxiv2026

Benthic biodiversity hotspots in the Weddell Sea: Bridging geomorphology, biogeography, and oceanography across scales

Benthic ecosystems are shaped by seafloor structure, yet linking geomorphology and biology across environmental gradients remains challenging. Here, we integrate seafloor imagery, multiscale bathymetry, and predictive modelling to quantify benthic biodiversity and its environmental drivers along the Powell Basin flank of the Antarctic Peninsula. Steep geomorphological landforms (terraces and steep slopes) hosted maximal densities and distinct communities, with significant enrichment of corals, sponges, ophiuroids, and sea pens. The marked congruence of slope across bathymetric resolutions enabled regional upscaling, estimating a standing stock of [~]96 billion individuals across 7,400 km{superscript 2} of the basin flank. Decadal oceanographic models indicate that benthic densities peak in cold bottom water below -0.15 {degrees}C. We identified four biodiversity hotspots ([~]33 km2) with slopes >35{degrees} and depths >1,700 m, with up to threefold higher densities. Hotspots align with the pathway of Weddell Sea Deep Water, where thermal decoupling between bottom and overlying waters indicates dynamic hydrography along steep, biodiverse terrain. Furthermore, contrasting sea-ice cover and stratification regimes suggest distinct cryo-pelagic-benthic coupling around hotspots. The concentration of biodiversity through seafloor geomorphology and ocean circulation bridges Antarctic benthic ecology from habitat to biogeography, with implications for monitoring change and guiding conservation in the warming Weddell Sea.

ecology↗

bioRxiv2026

Epigenetic control of microglial mitochondrial immunity by KAT7 drives Alzheimer's disease pathogenesis

Mitochondrial DNA (mtDNA)-driven innate immune signaling sustains chronic neuroinflammation in neurological diseases such as Alzheimers disease (AD), yet how this pathway is regulated in microglia remains poorly understood. Here, we identify the histone acetyltransferase KAT7 (HBO1) as a central epigenetic regulator that links chromatin remodeling to mitochondrial immune activation. KAT7 and its histone mark H3K14ac are elevated in microglia from 5xFAD mice and human AD brains. Integrative transcriptomic and epigenomic analyses reveal that KAT7 activates transcription of Cmpk2, a mitochondrial kinase essential for mtDNA synthesis. Loss of KAT7 reduces Cmpk2 expression, impairs mtDNA replication and release, and consequently suppresses cGAS-STING and NLRP3 signaling. Importantly, both microglia-specific deletion and pharmacological inhibition of KAT7 mitigate cytosolic mtDNA-induced neuroinflammation, decrease amyloid-{beta} burden, restore synaptic plasticity, and improve cognitive function in 5xFAD mice. Together, these findings uncover an epigenetic-mitochondrial axis sustaining microglial pathogenicity and establish KAT7 as a promising therapeutic target for AD.

neuroscience↗

bioRxiv2026

Iron Deficiency Drives Sarcopenia in the Elderly: HIF-1α-Mediated Fibro-Adipogenic Progenitor Differentiation Induces Fat Infiltration and Impairs Muscle Function

Abnormal iron metabolism is closely linked to sarcopenia; however, the specific iron metabolism features of fat-infiltrating sarcopenia remain poorly understood. Proteomic sequencing revealed that in skeletal muscle with severe fat infiltration, the expression of iron utilization-related proteins was significantly downregulated, whereas that of iron uptake and storage proteins was markedly upregulated, thereby presenting an abnormal iron deficiency (ID) phenotype. Nevertheless, the mechanism by which ID drives intramuscular fat infiltration has not been fully elucidated. Using clinical samples, aged murine ID models, and in vitro cell assays, this study is the first to demonstrate that ID stabilizes hypoxia-inducible factor-1 (HIF-1), promoting aberrant adipogenic differentiation of fibro-adipogenic progenitors (FAPs), disrupting the homeostatic balance between satellite cells (SCs) and FAPs, exacerbating skeletal muscle fat infiltration, and impairing muscle repair capacity. Notably, treatment with the HIF-1 inhibitor PX478 reversed these pathological alterations and improved muscle function. Collectively, our findings identify the ID-HIF-1-FAPs axis as a key driver of intramuscular fat infiltration, offering a novel therapeutic target for the clinical management of fat-infiltrating sarcopenia.

cell biology↗

bioRxiv2026

Nascent extracellular matrix converts biomaterial cues into cell fate decisions

Hydrogels serve as powerful models for investigating cell-extracellular matrix (ECM) interactions. While chemical modifications are routinely used to tune hydrogel properties, it remains unclear whether these modifications mediate cell fate. Previous work has shown that cells deposit newly synthesized (nascent) ECM at the cell-hydrogel interface. Here, we demonstrate that this nascent ECM interface regulates how cells interpret chemical modifications. Using hydrogels with varied chemical modifications, we isolated the effects of chemical modification on nascent ECM and cell fate. Nascent ECM deposition increased as a function of hydrogel modification and with distinct matrisome compositions. While low modification hydrogels promoted cell differentiation, high modifications increased cell proliferation. Perturbing cell-nascent ECM interactions reversed this cell fate. Our findings reveal that nascent ECM regulates cell fate by converting hydrogel cues into signals that control cell fate. This tri-directional interplay among hydrogel chemical modifications, nascent ECM, and cell fate reframes how we interpret cell-hydrogel interactions.

bioengineering↗

bioRxiv2025

PKU tags, novel genetically encoded shape tags for cell labeling in light and electron microscopy

Distinguishing among neuronal cell types is crucial for deciphering complex neural networks and brain functions. However, the current repertoire of cell-labeling tools compatible with light microscopy (LM) and/or electron microscopy (EM) is limited compared to the vast number of cell types in the brain. Here, we introduce PKU (polymer king-size unit) tags, genetically encoded "shape tags" that leverage the polymerization of self-assembling proteins, spectrally distinct fluorescent proteins and, optionally, a nuclear targeting sequence to generate a series of multi-shaped (spherical or filamentous), multi-colored (blue, green, red, near-infrared) and multi-localized (cytosolic or nuclear) tags. By co-expressing multiple PKU tags within the same cell, a combinatorial strategy further expands the repertoire, which can theoretically yield hundreds of unique labeling patterns. Expressing PKU tags in vivo provides multi-cell-type labeling and neuronal circuit tracing, without altering the animals behavior or transcriptomic profiles. Moreover, when fused to the peroxidase APEX2, PKU tags maintain their shape-specific features, providing shape "barcoding" using EM. Thus, PKU tags represent a versatile and efficient toolkit for studying connectomics using both LM and EM.

neuroscience↗

bioRxiv2025

Decorin promotes nascent proteoglycan retention in cartilage matrix by strengthening collagen II-aggrecan integration

Cartilage extracellular matrix (ECM), a hydrated collagen II-aggrecan composite, undergoes dynamic turnover during both normal homeostasis and disease-associated remodeling. This study elucidates a crucial role for decorin in promoting the retention and stability of nascent aggrecan within this matrix. By applying bio-orthogonal click-labeling, we demonstrate that loss of decorin accelerates the release of nascent aggrecan under both physiological and inflammatory conditions, without affecting its preferred localization to the pericellular matrix. Conversely, supplementation with exogenous decorin mitigates inflammation-induced loss of nascent aggrecan, supporting its potential as a therapeutic target. At the molecular level, decorin exhibits strong binding affinity for aggrecan, and enhances aggrecan-aggrecan and aggrecan-collagen II interactions, reinforcing its direct role in integrating cartilage matrix constituents. Also, by binding to collagen II, decorin stiffens the collagen II fibril network, thereby strengthening the confinement effect that limits the diffusive loss of entrapped aggrecan. Notably, decorin does not alter chondrocyte transcriptomic profiles in vivo, emphasizing its primary role in maintaining matrix integrity through biophysical mechanisms rather than cell signaling. Together, these findings provide a mechanistic foundation for developing decorin-based biomaterials or gene therapies aimed at preserving or regenerating the cartilage matrix for improved outcomes in osteoarthritis.

biophysics↗

bioRxiv2025

IGP receptors act as redox sensors to mediate broad-spectrum disease resistance induced by cellooligomers

Cys-oxidation is a well-established post-translational regulator of intracellular protein function, yet its role in extracellular receptor-mediated signaling remains poorly understood. Here, we investigate the LRR-Malectin receptor-like kinase IGP1, a key receptor for plant cell wall damage-associated molecular patterns (DAMPs). We demonstrate that IGP1 directly recognizes cellooligomers with a degree of polymerization [≥] 3 via a conserved binding groove in its LRR domain, not the malectin domain as anticipated. Strikingly, a pair of cysteines within the malectin domain functions as a redox sensor, undergoing hydrogen peroxide-induced oxidation that sterically reduces ligand binding affinity and attenuates immune signaling. Phylogenetic analyses reveal that IGP originated after the terrestrialization of plants, and is an evolutionarily conserved redox sensor in land plants. Functional studies in Arabidopsis (Arabidopsis thaliana), wheat (Triticum aestivum), and soybean (Glycine max) confirm that the IGP-cellooligomer module confers broad-spectrum disease resistance. Our work uncovers a unique mechanism where ligand perception and redox sensing are integrated into a single receptor to regulate DAMP-triggered immunity.

plant biology↗

bioRxiv2025

Construction, Phenotypic Characterization, and Immunomodulatory Function Study of BMSC-Macrophagy Hybrid in vitro

ObjectiveThe aim of our study was to integrate bone marrow-derived stem cells (BMSCs) with macrophages and to investigate the performance of this hybrid in IgG clearance, immunosuppression, and the mitigation of inflammatory injury. MethodsBMSCs and RAW264.7 cells were fused through transient transfection using the COVID-19 spike glycoprotein and human angiotensin-converting enzyme 2 (hACE2), respectively. The resulting hybrids were purified using magnetic sorting, and their characteristics were evaluated by examining morphology, glucose uptake rates, phagocytic capacity, cytoskeletal morphology, and the levels of Fc{gamma} receptors, as well as anti-inflammatory, antioxidant, adhesion, and complement inhibitory factors. The IgG clearance capability of the hybrids was assessed by measuring internalization efficiency and infiltration into renal organoids. Additionally, the reparative effects of the hybrids were evaluated using doxorubicin-treated MPC5 damage models. Live cell imaging techniques were employed to investigate the interactions between the hybrids and immature dendritic cells (iDCs). Hybrid-derived nanovesicles were isolated, and their abilities to target IgG and clear IL-6 were evaluated using immunofluorescence and ELISA. Furthermore, chloroplasts and C-dots were co-incubated with the hybrids, and their benefits were assessed by measuring cell viability, oxygen content, ROS levels, mitochondrial function, and the levels of anti-inflammatory, antioxidant, and pro-repair factors. A scratch assay was conducted to determine the impact of chloroplast transplantation on the migration ability of 3T3 cells. ResultsHigh-purity hybrids were successfully produced that cleared IgG while maintaining their anti-inflammatory and antioxidant effects. They promoted the recovery of podocyte injury through vesicular and mitochondrial delivery, as well as efferocytosis. Furthermore, the hybrids inhibited iDCs primarily through migrasomes, intercellular nanomicrotubule connections, and phagocytosis. The derived nanovesicles were capable of residing in IgG-enriched regions and adsorbing IL-6. Interestingly, the transplanted chloroplasts enabled the hybrids to utilize light energy to enhance their antioxidant capacity and promote the migration of 3T3 cells, which contributes to wound repair. Subsequently, the loading of C-dots was beneficial for enhancing resistance to oxidative damage. ConclusionsOur results suggest that hybrids-mediated therapy is a new and creative therapeutic approach for managing immune-mediated nephropathies.Hybrids demonstrated effective immunomodulatory and promoted injury recovery. The derived nanovesicles have the potential to alleviate the inflammatory burden at sites of antibody immune complex deposition. Additionally, the incorporation of chloroplasts and C-dots enhanced the hybrids adaptability to ischemic-hypoxic microenvironments.

cell biology↗

bioRxiv2025

Cryo-EM structure of plant urea transporter DUR3 reveals essential role of C-terminal domain in tetramer assembly and insights into proton-coupled transport

Nitrogen pollution, caused by the overuse of nitrogen-fertilizers such as urea in agricultural production, has become a serious problem that threatens both the environment and human health. Improving the nitrogen use efficiency of crops is an important approach to reduce fertilizer use. DUR3, a high-affinity urea transporter, enables plants to acquire urea from natural or agricultural soils with low urea concentrations and has been proposed as a promising target for engineering crops to improve nitrogen-fertilizer use efficiency. Despite extensive studies for more than 20 years, the structure, substrate recognition and transport mechanism of DUR3 have remained unknown. Here we report the cryo-EM structure of maize DUR3 (ZmDUR3) at 3.41 [A] resolution. ZmDUR3 adopts an amino acid-polyamine-organocation (APC) superfamily fold and is structurally distinct from animal facilitative urea transporters. Our structure provides important insights into urea recognition by DUR3 and suggests that a pair of acidic residues is potentially involved in proton coupling during urea transport. Furthermore, our structural and functional studies indicate that DUR3 assembles into a tetramer and tetramer formation is important for its function. Our results pave the way for engineering plant DUR3 protein for applications in sustainable agricultural production.

plant biology↗

bioRxiv2024

The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

COVID-19 (Coronavirus disease 19) is caused by infection with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in the respiratory system and other organ systems. Tissue injuries resulting from viral infection and host hyperinflammatory responses may lead to moderate to severe pneumonia, systemic complications, and even death. While anti-inflammatory agents have been used to treat patients with severe COVID-19, their therapeutic effects are limited. GPR4 (G protein-coupled receptor 4) is a pro-inflammatory receptor expressed on vascular endothelial cells, regulating leukocyte infiltration and inflammatory responses. In this study, we evaluated the effects of a GPR4 antagonist, NE-52-QQ57, in the SARS-CoV-2-infected K18-hACE2 transgenic mouse model. Our results demonstrated that GPR4 antagonist treatment increased the survival rate in this severe COVID-19 mouse model. The inflammatory response, characterized by proinflammatory cytokines and chemokines, was reduced in the GPR4 antagonist group compared with the vehicle group. Additionally, both SARS-CoV-2 RNA copy numbers and infectious viral titers in the mouse lung were decreased in the GPR4 antagonist group. The percentage of SARS-CoV-2 antigen-positive mouse brains was also decreased in the GPR4 antagonist group compared to the vehicle group. Furthermore, the GPR4 antagonist inhibited SARS-CoV-2 propagation in Vero E6 cells. Together, these results suggest that GPR4 antagonism may be explored as a novel approach for the treatment of COVID-19 and other similar viral diseases.

pharmacology and toxicology↗

bioRxiv2024

Ultrastructural membrane dynamics of mouse and human cortical synapses

Live human brain tissues provide unique opportunities for understanding synaptic transmission. Investigations have been limited to anatomy, electrophysiology, and protein localization--while crucial parameters such as synaptic vesicle dynamics were not visualized. Here we utilize zap-and-freeze time-resolved electron microscopy to overcome this hurdle. First, we validate the approach with acute mouse brain slices to demonstrate that slices can be stimulated to produce calcium signaling. Next, we show that synaptic vesicle endocytosis is induced in both mouse and human brain slices. Crucially, clathrin-free endocytic pits appear immediately next to the active zone, where ultrafast endocytosis normally occurs, and can be trapped at this location by a dynamin inhibitor. In both species a protein essential for ultrafast endocytosis, Dynamin 1xA, localizes to the region peripheral to the active zone, the putative endocytic zone, indicating a possible conserved mechanism between mouse and human. This approach has the potential to reveal dynamic, high-resolution information about synaptic membrane trafficking in intact human brain slices.

neuroscience↗