Biology subjects

Holcman, D.

Publications and source records attributed to Holcman, D..

At least 19 recordsLinked to original sources

bioRxiv2026

WNT/β-catenin increases 3D chromatin mobility and reorganisation during posterior cell state transitions

Chromatin mobility, the exploration of 3D conformations by the chromatin fibre, is implicated in transcription and DNA repair, and is thought to decrease progressively as cells differentiate. Here, combining live-cell 3D single-nucleosome tracking with genomic approaches, we show that chromatin mobility instead changes in a cell-type-specific manner, increasing as pluripotent cells acquire posterior fates. This is accompanied by 3D genome reorganisation, including loss of Polycomb-associated interactions and emergence of CDX2-associated interactions. Withdrawal of posteriorising WNT/{beta}-catenin signalling rapidly reduces chromatin mobility and partially restores Polycomb-associated chromatin interactions, while Cdx1/2/4-null cells fail to acquire these WNT/{beta}-catenin-dependent changes. Together, our findings establish chromatin mobility as a regulated property of cell state, with WNT/{beta}-catenin signalling and CDX factors acting as mediators that fine-tune the sampling of 3D chromatin interactions during cell state transitions.

cell biology↗

bioRxiv2026

Vesicular acidification modulates the synaptic current: a hybrid diffusion reaction model analysis

Glutamate synaptic vesicles co-release protons, producing a brief acidification of the synaptic cleft that could modulate AMPA receptor (AMPARs) operation. To evaluate the extent of receptor acidification, we develop a diffusion-reaction model that couples vesicle-evoked proton and glutamate transients to AMPAR dynamics. Our simulations reveal that the rapid diffusion of protons and glutamate within the flat-cylindrical synaptic cleft leads to a mixture of protonated, singly glutamate-bound and doubly glutamate-bound AMPARs. We studied four postsynaptic AMPAR distributions - uniform disk, sub-disk, Gaussian cluster, and point-like cluster - and showed a [~] 50% increase in the number of acidified receptors when AMPARs are clustered on the postsynaptic cleft compared to a uniform arrangement. We further explored the impact of pH revealing that at acidic conditions (pH [~] 5), approximately 80-90% of open receptors are non-acidified, whereas under strongly acidic conditions (pH [~] 3), about 80-90% of open receptors exist in the protonated form. Finally, we explored how acidification modulates AMPARs during paired-pulse stimulation, a measure of short-term synaptic depression. While the presence of protons does not markedly alter the overall trends, acidified receptor states reduce the occupancies of their neutral counterparts by roughly 10-15%, indicating a mild redistribution toward protonated receptor conformations. To conclude, our model suggests that AMPAR protonation can influence the synaptic current, and we predict that this effect is determined primarily by the dissociation kinetics of glutamate and protons from AMPAR.

neuroscience↗

bioRxiv2026

Synaptic Input Triggers On-Demand Spine-Specific Mitochondrial ATP Production and Delivery

Synaptic activity imposes acute energy demands, especially for restoring ionic gradients via pumps and exchangers that require ATP. While mitochondria are positioned near dendritic spines to meet this demand, how ATP is produced and delivered with spatial precision remains unclear. Here, using high-resolution calcium and ATP imaging, immuno-cytochemistry, and computational modeling, we demonstrate that synaptic input--but not back-propagating action potentials (bAPs)--triggers on-demand mitochondrial ATP production. This occurs only in spines containing a spine apparatus (SA), where calcium-induced calcium release (CICR) activates mitochondrial calcium uniporters (MCUs), initiating ATP synthesis. We show that ATP delivery is spatially constrained to mitochondrial regions facing the spine base, where ATP-synthase is enriched. Importantly, ATP produced elsewhere on the mitochondrial surface tends to diffuse into the dendrite. We further demonstrate that the delivery of ATP to the spine head is geometrically optimized: an intermediate spine neck length maximizes delivery efficiency. Mathematical modeling and simulations revealb that the time scale for ATP to reach and refill all head-localized exchangers is on the order of hundreds of milliseconds--fast enough to meet local metabolic needs. The present findings establish a mechanism in which nanoscale calcium signaling and mitochondrial architecture together ensure rapid, spatially targeted ATP delivery, tightly coupled to synaptic activity.

neuroscience↗

bioRxiv2026

Long-projection astrocytes challenge canonical territorial organization in the sleep-promoting VLPO

The ventrolateral preoptic nucleus (VLPO) is a key hypothalamic hub for non-rapid eye movement sleep, yet the glial architecture supporting its circuits remains poorly understood. Here, combining genetic labeling, high-resolution imaging and calcium imaging, we uncover unexpected astrocyte diversity in the VLPO. In addition to classical protoplasmic astrocytes, we identify paired "doublet" astrocytes associated with high local proliferative activity, as revealed by EdU incorporation. We further describe a population of long-projection astrocytes extending processes far beyond canonical astrocytic territories and contacting distant cells. These projections challenge the classical territorial organization of astrocytes and resemble morphologies previously thought to be restricted to hominid brains. Notably, VLPO astrocytes display robust spontaneous Ca{superscript 2} activity and a highly functionally connected network compared to astrocytes in the cortex and hippocampus. Together, these findings reveal specialized astrocyte architectures and enhanced glial network integration within a sleep-promoting nucleus. Reporting summaryBellier et al. identify three astrocyte subtypes in the sleep-promoting VLPO, including long-projection astrocytes with hominid-like morphology. They uncover marked postnatal gliogenesis, distinctive spontaneous Ca{superscript 2} dynamics, and tightly interconnected astrocytic networks, revealing region-specific astrocyte specialization and enhanced glial communication.

neuroscience↗

bioRxiv2025

Thalamocortical coupling and cortical E-I balance generate diverse anesthetic α-spindles for interpretable EEG decoding

The neuronal circuits generating frequency (8-13Hz) oscillatory patterns during anesthesia are poorly understood, making their use difficult in predictive medicine. Here, we combine large-scale in vivo recordings of thalamocortical neuronal ensembles in anesthetized mice with a minimal neural mass model. We report non-rhythmic, weakly synchronized firing of thalamic and cortical neurons correlated with oscillatory patterns, indicating an emergent network rather than cellular dynamics. The associated imbalance between the firing of excitatory and inhibitory cortical neurons, together with an increase in firing fluctuations, favors larger amplitudes oscillations, whereas changes in thalamic neuron output control oscillation frequency. A neural mass model, comprising reciprocally connected excitatory and inhibitory cortical and thalamic neurons well reproduces the oscillation patterns observed in vivo at increasing anesthetic doses. These results provide a direct link between EEG oscillations during anesthesia and their underlying network/cellular dynamics which are well captured by a computational model that is clinically relevant for anesthetized brain state monitoring. HighlightsO_LI transient patterns during anesthesia emerge from thalamocortical and cortical excitatory-inhibitory (E-I) network dynamics. C_LIO_LI pattern frequency and amplitude reflect depth of anesthesia, dynamic thalamocortical equilibrium and E-I imbalance. C_LIO_LIEmergence of spindle-like patterns at multiple frequency defines newly characterized brain-state during anesthesia C_LIO_LIA mass-model reproduces the transient nature and spectral diversity of anesthetic-induced oscillations, linking neural circuit mechanisms to EEG output. C_LI

neuroscience↗

bioRxiv2025

Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

Maintenance of naive pluripotency requires core transcription factors (TFs) like SOX2 and auxiliary TFs like NANOG, yet molecular mechanisms governing their intra-nuclear dynamics and DNA binding interactions remain unclear. Here, using high-density 3D single-molecule light-field microscopy combined with novel spatiotemporal analysis pipelines, we track SOX2 and NANOG dynamics in live cells. Despite lower protein abundance, NANOG displays a similar chromatin-bound fraction to SOX2. This arises partially because, while both TFs undergo frequent transient non-specific binding interactions ([~]0.5-0.7s), NANOG exhibits more stable specific binding ([~]25s vs [~]16s). Both TFs also assemble into phase-separated domains of [~]400 nm containing both freely diffusing and chromatin-bound proteins, which further influences their dynamics. Strikingly, NANOGs protein-protein interaction domain markedly increases chromatin residence time (>5-fold) and the size of these phase-separated domains. Our work uncovers how NANOG and SOX2 stabilise gene regulatory networks that maintain naive pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics.

cell biology↗

bioRxiv2025

Column-Like Subnetwork Reconstruction in Motor Cortex from Graph-Based 3D High-Density Two-Photon Calcium Imaging

How precise 3D interactions among cortical neurons underlie layer-specific computations remains elusive. We developed a graph-framework to infer functional connectivity from fast volumetric two-photon Ca2+ imaging in the awake mouse primary motor cortex. By converting deconvolved traces into binary spike trains, removing population bursts, and applying an adaptive, layer-specific statistical threshold, we reconstructed a directed, weighted network of [~]1,000 neurons. Decomposition into strongly connected components revealed [~]10-cell subnetworks, predominantly in layer II/III but often bridging to layer Va. Across six 20-min recordings, we found that (1) layer II/III dominates connectivity, (2) feedback (Va[->]II/III) links are more numerous and stronger than the feed-forward (II/III[->]Va) ones, and (3) information flows in [≤] 6 synapses. We uncovered seven geometrical and dynamical motifs--ranging from compact columns to elongated diagonals--each with characteristic event sizes and durations. These findings reveal diverse, column-like microcircuits in M1 with a net ascending flow, suggesting that such subnetworks form elemental processing modules for motor control.

neuroscience↗

bioRxiv2025

A non-catalytic role for MLL2 in controlling chromatin organisation and mobility during the priming of pluripotent cells for differentiation

How the chromatin regulator MLL2 (KMT2B) influences cell differentiation remains poorly understood. MLL2 is the main histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for ESCs to differentiate into neuroectoderm. We show here that this requirement occurs during exit from naive pluripotency, days before neuroectoderm differentiation is impaired. Although MLL2 knockout has only a subtle effect on transcription during exit, reducing the expression of a few important neuroectodermal transcription factors, it substantially remodels chromatin architecture, disrupting 3D chromatin loops associated with bivalent promoters. The enzymatic activity of MLL2 is not needed for stabilising these loops or for neuroectoderm differentiation. This non-catalytic function of MLL2 in stabilising 3D chromatin loops has implications for lineage specification. Because MLL2 shares features with all four MLLs, chromatin tethering, rather than H3K4 methylation, may represent the primary function of MLL proteins during lineage commitment.

cell biology↗

bioRxiv2024

Insulation between adjacent TADs is controlled by the width of their boundaries through distinct mechanisms

Topologically Associating Domains (TADs) are sub-Megabase regions in vertebrate genomes with enriched intra-domain interactions that restrict enhancer-promoter contacts across their boundaries. However, the mechanisms that separate TADs remain incompletely understood. Most boundaries between TADs contain CTCF binding sites (CBSs), which individually contribute to the blocking of Cohesin-mediated loop extrusion. Using genome-wide classification, we show here that TAD boundary width forms a continuum from narrow to highly extended and correlates with CBS distribution, chromatin features, and gene regulatory elements. To investigate how these boundary widths emerge, we modified the Random Cross-Linker (RCL) polymer model to incorporate specific boundary configurations, enabling us to evaluate the differential impact of boundary composition on TAD insulation. Our analysis identifies three generic boundary categories, each influencing TAD insulation differently, with varying local and distal effects on neighboring domains. Notably, we find that increasing boundary width reduces long-range inter-TAD contacts, as confirmed by Hi-C data. While blocking loop extrusion at boundaries indirectly promotes spurious intermingling of neighboring TADs, extended boundaries counteract this effect, emphasizing their role in maintaining genome structure. In conclusion, TAD boundary width not only enhances the efficiency of loop extrusion blocking but may also modulate enhancer-promoter contacts over long distances across TADs boundaries, providing a mechanism for transcriptional regulation. Significance statementTopologically Associating Domains (TADs) compartmentalize vertebrate genomes to limit cross-domain enhancer-promoter loops. Our study reveals that boundaries between TADs are diverse genomic entities that range from narrow to highly extended. Using an interdisciplinary approach that combines genome-wide data analysis with biophysical polymer modeling, we find that wider TAD boundaries reduce spurious long-range interactions between neighboring domains. Moreover, we reveal how different boundary components can create this difference in insulating capacity. Our identification and characterization of TAD boundary width and composition suggests they have the potential to regulate the formation of enhancer-promoter loops across TAD boundaries at close and long distance.

genomics↗

bioRxiv2024

Unveiling the Functional Connectivity of Astrocytic Networks with AstroNet, a Graph Reconstruction Algorithm Coupled to Image Processing

Astrocytes form extended intercellular networks, displaying complex calcium activity. However, the specific organization of these astrocytic networks and the precise extent of their functional connectivity in different brain areas remain unexplored. To unveil the functional architecture of astrocytic networks, we developed, using a data-driven methodology, a novel algorithm called AstroNet that uses two-photon calcium imaging to map temporal correlations in activation events among neighboring astro-cytes. Our approach involves reconstructing functional astrocytic networks by organizing individual astrocyte activation events chronologically. This chronological order creates activity paths that enable the extraction of local astrocyte functional correlations. Ultimately, by tallying the occurrences of direct co-activations between pairs of cells along these pathways, we construct a graph that mirrors the underlying astrocyte functional network. By applying this method to two distinct brain regions (CA1 hippocampus and motor cortex), we identified notable differences in local network organizations in sub-regions of around 20-40 astrocytes. Specifically, the cortex exhibited a lower connectivity, while astrocytes in the hippocampus displayed stronger connections. Moreover, we found that in both regions, astrocytic networks consist of smaller, tightly connected sub-networks embedded within a larger, more loosely connected one. Altogether, our innovative method enables the identification of activation paths among astrocytes, facilitates the characterization of local network functional connectivity, and quantifies distinct connectivity patterns among astrocytes from different brain regions. This approach sheds light on the heterogeneous functional organization of astrocytic networks within the brain, pointing to region-specific astrocyte connectivity.

neuroscience↗

bioRxiv2023

Analyzing photoactivation with diffusion models to study transport in the Endoplasmic Reticulum network

Photoactivation is a paradigm consisting in local molecular fluorescent activation by laser illumination in a chosen region (source) while measuring the concentration at a target region. Data-driven modeling is concern with the following questions: how from the measurement in these two regions, is it possible to infer the properties of molecular propagation? How is it possible to use such reponses to infer motions occurring in networks such as the endoplasmic reticulum? In this article, we present a data-driven analysis based on diffusion-transport models and numerical simulations to interpret the photoactivation dynamics and extract biophysical parameters. Finally we discuss modeling approaches to reconstruct local network properties from photoactivation transients.

cell biology↗

bioRxiv2023

Mapping general anesthesia states based on electro-encephalogram transition phases

Cortical electro-encephalography (EEG) has become the clinical reference for monitoring unconsciousness during general anesthesia. The current EEG-based monitors classify general anesthesia states simply as underdosed, adequate, or overdosed, with no transition phases among these states, and therefore no predictive power. To address the issue of transition phases, we analyzed EEG signal of isoflurane-induced general anesthesia in mice. We adopted a data-driven approach and utilized signal processing to track{theta} - and{delta} - band dynamics as well as iso-electric suppressions. By combining this approach with machine learning, we developed a fully-automated algorithm. We found that the dampening of the{delta} -band occurred several minutes before significant iso-electric suppression episodes. Additionally, we observed a distinct{gamma} -frequency oscillation that persisted for several minutes during the recovery phase following isoflurane-induced overdose. Finally, we constructed a map summarizing multiple states and their transitions which can be utilized to predict and prevent overdose during general anesthesia. The transition phases we identified and algorithm we developed may allow clinicians to prevent inadequate anesthesia, and thus individually tailor anesthetic regimens. 1 Significance statementIn human patients, overdosing during general anesthesia can lead to cognitive impairment. Cortical electro-encephalograms are used to measure the depth of anesthesia. They allow for correction, but not prevention, of overdose. However, data-driven approaches open new possibilities to predict the depth of anesthesia. We established an electro-encephalogram signalprocessing pipeline, and constructed a predictive map representing an ensemble of gradual sedation states during general anesthesia in mice. In particular, we identified key electroencephalogram patterns which anticipate signs of overdose several minutes before they occur. Our results bring a novel paradigm to the medical community, allowing for the development of individually tailored and predictive anesthetic regimens.

neuroscience↗

bioRxiv2023

Simultaneous photoactivation and high-speed structural tracking reveal diffusion-dominated motion in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a structurally complex, membrane-enclosed compartment that stretches from the nuclear envelope to the extreme periphery of eukaryotic cells. The organelle is crucial for numerous distinct cellular processes, but how these processes are spatially regulated within the structure is unclear. Traditional imaging-based approaches to understanding protein dynamics within the organelle are limited by the convoluted structure and rapid movement of molecular components. Here, we introduce a combinatorial imaging and machine learning-assisted image analysis approach to track the motion of photoactivated proteins within the ER of live cells. We find that simultaneous knowledge of the underlying ER structure is required to accurately analyze fluorescently-tagged protein redistribution, and after appropriate structural calibration we see all proteins assayed show signatures of Brownian diffusion-dominated motion over micron spatial scales. Remarkably, we find that in some cells the ER structure can be explored in a highly asymmetric manner, likely as a result of uneven connectivity within the organelle. This remains true independently of the size or folding state of the fluorescently-tagged molecules, suggesting a potential role for ER connectivity in driving spatially regulated biology in eukaryotes.

cell biology↗

bioRxiv2023

High changing curvature regions detect chromatin enrichment in single cell data

Chromatin organization is nowadays accessible from population and single cell Hi-C data in the form of large contact matrices. Yet reconstructing the three-dimensional chromatin structure remains challenging and relies on polymer models and numerical simulations to account for these Hi-C data. Here we propose a novel optimization algorithm to identify cross-linker ensemble reproducing the experimental contact matrix. Furthermore, based on the polymer configurations extracted from the optimization procedure, we identify high changing curvature (HCC) regions in the chromatin, detected both in population and in single cell Hi-C, that we also compare to Topological Associated Domains (TADs). We report here that the HCC regions correlate with gene expression and CTCF high density distribution. Interestingly, the HCC region locations are heterogeneous across one cell repeats, revealing cell-to-cell variability. To conclude, HCC regions appear both in single and population Hi-C polymer reconstruction and can provide a possible unit for gene regulation.

genomics↗

bioRxiv2023

Experiments in micro-patterned model membranes support the narrow escape theory

The narrow escape theory (NET) predicts the escape time distribution of Brownian particles confined to a domain with reflecting borders except for one small window. Applications include molecular activation events in cell biology and biophysics. Specifically, the mean first passage time [Formula] can be analytically calculated from the size of the domain, the escape window, and the diffusion coefficient of the particles. In this study, we systematically tested the NET in a disc by variation of the escape opening. Our model system consisted of micro-patterned lipid bilayers. For the measurement of [Formula], we imaged diffusing fluorescently-labeled lipids using single-molecule fluorescence microscopy. We overcame the lifetime limitation of fluorescent probes by re-scaling the measured time with the fraction of escaped particles. Experiments were complemented by matching stochastic numerical simulations. To conclude, we confirmed the NET prediction in vitro and in silico for the disc geometry in the limit of small escape openings. Significance StatementIn the biological context of a cell, a multitude of reactions are facilitated by diffusion. It is astonishing how Brownian motion as a cost-efficient but random process is mediating especially fast reactions. The formalism of the narrow escape theory is a tool to determine the average timescale of such processes to be completed (mean first passage time, MFPT) from the reaction space and diffusion coefficient. We present the systematic proof of this formalism experimentally in a bio-mimetic model system and by random walk simulations. Further, we demonstrate a straightforward solution to determine the MFPT from incomplete experimental traces. This will be beneficial for measurements of the MFPT, reliant on fluorescent probes, that have prior been inaccessible.

biophysics↗

bioRxiv2022

Astroglial gap junctions strengthen hippocampal network activity by sustaining afterhyperpolarization via KCNQ channels

Throughout the brain, astrocytes form networks mediated by gap-junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecularly gap junction channels shape neuronal network patterns remains unclear. Here using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we found that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a novel facilitation-depression model, we identified neuronal afterhyperpolarization as the key parameter underlying bursting patterns regulation by extracellular potassium in mice with disconnected astrocytes. We confirmed experimentally this prediction, and revealed that astroglial network-control of extracellular potassium sustains neuronal afterhyperpolarization via activation of KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap-junctions mechanistically strengthen neuronal population bursts, and points to approaches for controlling aberrant activity in neurological diseases.

neuroscience↗

bioRxiv2021

High-throughput super-resolution single particle trajectory analysis reconstructs organelle dynamics and membrane re-organization

Super-resolution imaging can generate thousands of single-particle trajectories. These data can potentially reconstruct subcellular organization and dynamics, as well as measure disease-linked changes. However, computational methods that can derive quantitative information from such massive datasets are currently lacking. Here we present data analysis and algorithms that are broadly applicable to reveal local binding and trafficking interactions and organization of dynamic sub-cellular sites. We applied this analysis to the endoplasmic reticulum and neuronal membrane. The method is based on spatio-temporal time window segmentation that explores data at multiple levels and detects the architecture and boundaries of high density regions in areas that are hundreds of nanometers. By statistical analysis of a large number of datapoints, the present method allows measurements of nano-region stability. By connecting highly dense regions, we reconstructed the network topology of the ER, as well as molecular flow redistribution, and the local space explored by trajectories. Segmenting trajectories at appropriate scales extracts confined trajectories, allowing quantification of dynamic interactions between lysosomes and the ER. A final step of the method reveals the motion of trajectories relative to the ensemble, allowing reconstruction of dynamics in normal ER and the atlastin-null mutant. Our approach allows users to track previously inaccessible large scale dynamics at high resolution from massive datasets. The SPtsAnalysis algorithm is available as an ImageJ plugin that can be applied by users to large datasets of overlapping trajectories and offer a standard of SPTs metrics.

cell biology↗

bioRxiv2021

Emergence and fragmentation of the alpha-band driven by neuronal network dynamics

Rhythmic neuronal network activity underlies brain oscillations. To investigate how connected neuronal networks contribute to the emergence of the -band and the regulation of Up and Down states, we study a model based on synaptic short-term depression-facilitation with afterhyperpolarization (AHP). We found that the -band is generated by the network behavior near the attractor of the Up-state. Coupling inhibitory and excitatory networks by reciprocal connections leads to the emergence of a stable -band during the Up states, as reflected in the spectrogram. To better characterize the emergence and stability of thalamocortical oscillations containing and{delta} rhythms during anesthesia, we model the interaction of two excitatory with one inhibitory networks, showing that this minimal network topology leads to a persistent -band in the neuronal voltage characterized by dominant Up over Down states. Finally, we show that the emergence of the -band appears when external inputs are suppressed, while the fragmentation occurs at small synaptic noise or with increasing inhibitory inputs. To conclude, interaction between excitatory neuronal networks with and without AHP seems to be a general principle underlying changes in network oscillations that could apply to other rhythms. Author summaryBrain oscillations recorded from electroencephalograms characterize behaviors such as sleep, wakefulness, brain evoked responses, coma or anesthesia. The underlying rhythms for these oscillations are associated at a neuronal population level to fluctuations of the membrane potential between Up (depolarized) and Down (hyperpolarized) states. During anesthesia with propofol, a dominant alpha-band (8-12Hz) can emerge or disappear, but the underlying mechanisms remain unclear. Using modeling, we report that the alpha-band appears during Up states in neuronal populations driven by short-term synaptic plasticity and noise. Moreover, we show that three connected networks representing the thalamocortical loop reproduce the dynamics of the alpha-band, which emerges following the arrest of excitatory stimulations, but can disappear by increasing inhibitory inputs. To conclude, short-term plasticity in well connected neuronal networks can explain the emergence and fragmentation of the alpha-band.

neuroscience↗