Biology subjects

Holcman, D.

Publications and source records attributed to Holcman, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

A complex CTCF binding code defines TAD boundary structure and function

Topologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair1-10. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundaries11-20. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary21-24. Here, we report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals that this clustering of CTCF binding does not only occur among peaks but also frequently within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites additively contribute to TAD separation. This clustering of CTCF binding may counter against the dynamic DNA-binding kinetics of CTCF25-27, which urges a re-evaluation of current models for the blocking of loop extrusion21-23. Our work thus reveals an unanticipatedly complex code of CTCF binding at TAD boundaries that expands the regulatory potential for TAD structure and function and can help to explain how distant non-coding structural variation influences gene regulation, DNA replication, recombination and repair5,28-34.

genetics↗

bioRxiv2020

Chromatin memory generated by cohesin-like interaction at looping sites

Chromatin loops inside the nucleus can be stable for a very long time, which remains poorly understood. Such a time is crucial for chromatin organization maintenance and stability. We explore here several physical scenarios, where loop maintenance is due to diffusing cross-linkers such as cohesin and CTCF that can bind and unbind at the base of chromatin loops. Using a Markov chain approach to coarse-grain the binding and unbinding, we consider that a stable loop disappears when the last cross-linker (CTCF or cohesin molecule) is unbound. We derive expressions for this last passage times that we use to quantify the loop stability for various value parameters, such as the chemical rate constant or the number of cross-linkers. The present analysis suggests that this binding and unbinding mechanism is sufficient to guarantee that there are always cross-linkers in place because they generate a positive feed-back mechanism that stabilizes the loop over long-time. To conclude, we propose that tens to hundreds cross-linkers per loop are sufficient to guarantee the loop stability in the genome over a cell cycle.

genetics↗

bioRxiv2020

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.

cell biology↗

bioRxiv2020

The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes

The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains, sheets and interconnected tubules. These domains undergo dynamic reshaping, in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling within minutes are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status. The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven re-positioning of lysosomes, which leads to both a redistribution of the ER tubules and its global morphology. Lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.

cell biology↗

bioRxiv2019

Multiple activities of the MatP protein are involved in post-replicative pairing of sister chromosomes in Escherichia coli

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non-ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Chemotaxis and topotaxis add vectorially for amoeboid cell migration

Cells encounter a wide variety of physical and chemical cues when navigating their native environments. However, their response to multiple simultaneous cues is not yet clear. In particular, the influence of topography, in the presence of a chemotactic gradient, on their migratory behavior is understudied. Here, we investigate the effects of topographical guidance on highly motile amoeboid cell migration (topotaxis) generated by asymmetrically placed micropillars. The micropillar field allows for an additional, natural chemotactic gradient in two different directions, thereby revealing the relevance of topotaxis in the presence of cell migration directed by chemical gradients (chemotaxis). Interestingly, we found that the topotactic drift generated by the pillar field is conserved during chemotaxis. We show that the drifts generated by both these cues add up linearly. A coarse-grained analysis as a function of pillar spacing subsequently revealed that the strength and direction of the topotactic drift is determined by (i) the pore size, (ii) space between pores, and (iii) the effective diffusion constant of the cells. Finally, we argue that topotaxis must be conserved during chemotaxis, as it is an emergent property of both the asymmetric properties of the pillar field and the inherent stochasticity of (biased) amoeboid migration.

biophysics↗

bioRxiv2019

Telomeric chromosome ends are highly mobile and behave like free double-strand DNA breaks

Chromosome organization and dynamics are critical for DNA transactions, including gene expression, replication, and DNA repair. In yeast, the chromosomes are anchored through their centromeres to the spindle pole body, and their telomeres are grouped into clusters at the nuclear periphery, constraining chromosome mobility. Here, we have used experimental and computational approaches to study the effects of chromosome-nuclear envelope (NE) attachments on the dynamics of S. cerevisiae chromosomes. We found that although centromere proximal loci were, as predicted, more dynamically constrained than distal loci, telomeres were highly mobile, even when positioned at the nuclear periphery. Polymer modeling indicated that polymer ends are intrinsically more mobile than internal sites. We tested this model by measuring the mobility of a double strand break (DSB) end within a chromosome arm. Upon separation of the DSB ends, their mobility significantly increased. Altogether, our results reveal that telomeres behave as highly mobile polymer ends, despite interactions with the nuclear membrane.

cell biology↗

bioRxiv2019

Predictive analysis of patient recovery from cardiac-respiratory arrest

The severity of neuronal damages in comatose patients following anoxic brain injury can be probed by evoked auditory responses. However, it remains challenging to predict the return to full consciousness of post-anoxic coma of hospitalized patients. We presented here a method to predict the return to consciousness based on the analysis of periodic responses to auditory stimulations, recorded from surface cranial electrodes. The input data are event-related potentials (ERPs), recorded non-invasively with electro-encephalography (EEG). We extracted several novel features from the time series responses in a window of few hundreds of milliseconds from deviant and non-deviant auditory stimulations. We use these features to construct two-dimensional statistical maps, that show two separated clusters for recovered (conscience) and deceased patients, leading to a high classification success as tested by a cross-validation procedure. Finally, using Gaussian, K-neighborhood and SVM classifiers, we construct probabilistic maps to predict the outcome of post-anoxic coma. To conclude, statistics of deviant and non-deviant responses considered separately provide complementary and confirmatory predictions for the outcome of anoxic coma.

neuroscience↗

bioRxiv2019

Reconstructing wells from high density regions extracted from super-resolution single particle trajectories

Large amount of super-resolution single particle trajectories has revealed that the cellular environment is enriched in heterogenous regions of high density, which remain unexplained. The biophysical properties of these regions are characterized by a drift and their extension (a basin of attraction) that can be estimated from an ensemble of trajectories. We develop here two statistical methods to recover the dynamics and local potential wells (field of force and boundary) using as a model a truncated Ornstein-Ulhenbeck process. The first method uses the empirical distribution of points, which differs inside and outside the potential well, while the second focuses on recovering the drift field. Finally, we apply these two methods to voltage-gated calcium channels and phospholipids moving on the surface of neuronal cells and recover the energy and size of these high-density regions with nanometer precision.

biophysics↗

bioRxiv2019

Single particle trajectory statistic to reconstruct chromatin organization and dynamics

Chromatin organization remains complex and far from understood. We discuss here recent statistical methods to extract biophysical parameters from in vivo single particle trajectories of loci to reconstruct chromatin reorganization in response to cellular stress such as DNA damages. We look at the methods to analyze both single loci as well as multiple loci tracked simultaneously and explain how to quantify and describe chromatin motion using a combination of extractable parameters. These parameters can be converted into information about chromatin dynamics and function. Furthermore, we discuss how the time scale of recurrent motion of a locus can be extracted and converted into local chromatin dynamics. We also discuss the effect of various sampling rates on the estimated parameters. Finally, we discuss polymer methods based on cross-linkers that account for minimal loop constraints hidden in tracked loci, that reveal chromatin organization at the 250nm spatial scale. We list and refer to some algorithm packages that are now publicly available. To conclude, chromatin organization and dynamics at hundreds of nanometers can be reconstructed from locus trajectories and predicted based on polymer models.

genomics↗

bioRxiv2018

Local decondensation at double-stranded DNA breaks modifies chromatin at long distances and reduces encounter times during homology search

Double-strand break (DSB) repair by homologous recombination (HR) requires an efficient and timely search for a homologous template. Here we first study global chromatin re-organization following a single DSB: due to the potential release of cross-linkers such as cohesin and CTCF molecules near the DSB site, loops are released and chromatin is decondensed, explaining the change of chromatin locus motion at larger genomic distances. This mechanism provides an elementary explanation for the increase of the anomalous exponent at sites located far away from the DSB, after break induction. Second, we explore the consequences of chromatin reorganization for the homology search during DNA repair: using polymer models, we estimate the mean first encounter time (MFET) between two loci on the chromatin in a confined nucleus. Reducing tethering forces, as reported experimentally on chromatin, is associated with a local de-condensation near the break followed by the extrusion of the breaks. Consequently, we report here that the mean first encounter time between homologous sites is decreased by two orders of magnitude even when the homologue sequence is located on the nuclear boundary. To conclude, our results suggest that local changes in inter-nucleosomal contacts near DSBs, by cohesin removal, remodel the chromatin and drastically shorten the time required to complete a long-range search for a homologous template.

genetics↗

bioRxiv2018

The first one hundred nanometers inside the pre-synaptic terminal where calcium diffusion triggers vesicular release

Calcium diffusion in the thin one hundred nanometers layer located between the plasma membrane and docked vesicles in the pre-synaptic terminal of neuronal cells mediates vesicular fusion and synaptic transmission. Accounting for the narrow-cusp geometry located underneath the vesicle is a key ingredient that defines the probability and the time scale of calcium diffusion to bind calcium sensors for the initiation of vesicular release. We study here the time scale, the calcium binding dynamics and the consequences for asynchronous versus synchronous release. To conclude, threedimensional modeling approaches and the associated coarse-grained simulations can now account efficiently for the precise co-organization of vesicles and Voltage-Gated-Calcium-Channel (VGCC). This co-organization is a key determinant of short-term plasticity and it shapes asynchronous release. Moreover, changing the location of VGCC from few nanometers underneath the vesicle modifies significantly the release probability. Finally, by modifying the calcium buffer concentration, a single synapse can switch from facilitation to depression.

biophysics↗

bioRxiv2018

Dynamics of ion fluxes between neurons, astrocytes and the extracellular space during neurotransmission

Ionic homeostasis in the brain involves redistribution of ionic fluxes in several cell types and compartments, including neurons, astrocytes and the extracellular space. How the major ionic activity-dependent fluxes of potassium and sodium are individually regulated remains difficult to dissociate and to track experimentally. We here review recent progress in modeling the ionic fluxes evoked by neuronal activity based on mass conservation. Excitability of neurons indeed relies on inward sodium and outward potassium fluxes during action potential firing. Recently, we have developed a tri-compartment model based on mass-action kinetics equations that account for potassium dynamics between neurons, astrocytes and the extracellular space. This review describes how such type of model can be used to spatially and temporally predict potassium fluxes during various regimes of neuronal activity. In particular, the model initially showed that it takes several seconds for astrocytes to buffer the majority of the potassium rapidly released by neurons in both basal and high regime of activity. Such model can also probe the selective contribution of ionic channels, and revealed for instance that disruption of the main astroglial potassium Kir4.1 channels not only favors the emergence of epileptiform activity, but also dysregulates neuronal excitability specifically during slow rhythmic activities. We here also extend the predictions of the model by assessing the selective contribution of the astroglial and neuronal Na/K ATPase, or volume of the extracellular space on potassium dynamics. We discuss these findings and their implications for neuronal information processing in the healthy and diseased brain.

neuroscience↗

bioRxiv2018

Fast calcium transients in neuronal spines is driven by extreme statistics

Extreme statistics describe the distribution of rare events that can define the timescales of transduction within cellular microdomains. We combine biophysical modeling and analysis of live-cell calcium imaging to explain the fast calcium transient in spines. We show that in the presence of a spine apparatus (SA), which is an extension of the smooth endoplasmic reticulum (ER), calcium transients during synaptic inputs rely on rare and extreme calcium ion trajectories. Using numerical simulations, we predicted the asymmetrical distributions of Ryanodine receptors and SERCA pumps that we confirmed experimentally. When calcium ions are released in the spine head, the fastest ions arriving at the base determine the transient timescale through a calcium-induced calcium release mechanism. In general, the fastest particles arriving at a small target are likely to be a generic mechanism that determines the timescale of molecular transduction in cellular neuroscience.\n\nSignificance statementIntrigued by fast calcium transients of few milliseconds in dendritic spines, we investigated its underlying biophysical mechanism. We show here that it is generated by the diffusion of the fastest calcium ions when the spine contains a Spine Apparatus, an extension of the endoplasmic reticulum. This timescale is modulated by the initial number of released calcium ions and the asymmetric distribution of its associated calcium release associated Ryanodyne receptors, present only at the base of a spine. This novel mechanism of calcium signaling that we have unraveled here is driven by the fastest particles. To conclude, the rate of arrival of the fastest particles (ions) to a small target receptor defines the timescale of activation instead of the classical forward rate of chemical reactions introduced by von Smoluchowski in 1916. Applying this new rate theory to transduction should refine our understanding of the biophysical mechanisms underlying molecular signaling.

cell biology↗

bioRxiv2018

Electrical transient laws in neuronal microdomains based on electro-diffusion

The current-voltage (I-V) conversion characterizes the physiology of cellular microdomains and reflects cellular communication, excitability, and electrical transduction. Yet deriving such I-V laws remains a major challenge in most cellular microdomains due to their small sizes and the difficulty of accessing voltage with a high nanometer precision. We present here novel analytical relations derived for different numbers of ionic species inside a neuronal micro/nano-domains, such as dendritic spines. When a steady-state current is injected, we find a large deviation from the classical Ohms law, showing that the spine neck resistance is insuficent to characterize electrical properties. For a constricted spine neck, modeled by a hyperboloid, we obtain a new I-V law that illustrates the consequences of narrow passages on electrical conduction. Finally, during a fast current transient, the local voltage is modulated by the distance between activated voltage-gated channels. To conclude, electro-diffusion laws can now be used to interpret voltage distribution in neuronal microdomains.

neuroscience↗

bioRxiv2017

Heterogeneous cross-linked polymers to reconstruct chromatin reorganization during cell differentiation

Chromatin of mammalian nucleus folds into discrete contact enriched regions such as Topologically Associating domains (TADs). The folding hierarchy of the TADs and their internal organization is highly dynamic through cellular differentiation, where structural changes within and between TADs are correlated with gene activation and silencing. To account for multiple interacting TADs, we developed a parsimonious randomly cross-linked (RCL) polymer model that maps high frequency encounters present in HiC data within and between TADs into direct local monomer interactions, characterized by the number of cross-links at a given base-pair resolution. We reconstruct three TADs obtained from the mammalian X chromosome for three stages of differentiation. We compute the radius of gyration of TADs and the encounter probability between genomic segments. We found 1) a synchronous compaction and decompaction of TADs throughout differentiation and 2) secondary structures such as meta-TADs in 5C data resulting from weak inter-TAD interactions. Finally, the present method links steady-state to dynamic properties of the chromatin revealed by the distribution of anomalous exponents of single loci trajectories, reconstructed from HiC data.

genomics↗

bioRxiv2017

Redundancy principle for optimal random search in biology

Chemical activation rate is traditionally determined by the diffusion flux into an absorbing ball, as computed by Smoluchowski in 1916. Thus the rate is set by the mean first passage time (MFPT) of a Brownian particle to a small target. This paradigm is shifted in this manuscript to set the time scale of activation in cellular biology to the mean time of the first among many arrivals of particles at the activation site. This rate is very different from the MFPT and depends on different geometrical parameters. The shift calls for the reconsideration of physical modeling such as deterministic and stochastic chemical reactions based on the traditional forward rate, especially for fast activation processes occurring in living cells. Consequently, the biological activation time is not necessarily exponential. The new paradigm clarifies the role of population redundancy in accelerating search processes and in defining cellular-activation time scales. This is the case, for example, in cellular transduction or in the nonlinear dependence of fertilization rate on the number of spermatozoa. We conclude that statistics of the extreme set the new laws of biology, which can be very different from the physical laws derived for individuals.

biophysics↗

bioRxiv2017

Two Loci Single Particle Trajectories Analysis: Constructing A First Passage Time Statistics Of Local Chromatin Exploration

Stochastic single particle trajectories are used to explore the local chromatin organization. We present here a statistical analysis of the first contact time distributions between two tagged loci recorded experimentally. First, we extract the association and dissociation times from data for various genomic distances between loci and we show that the looping time occurs in confined nanometer regions. Second, we characterize the looping time distribution for two loci in the presence of multiple DNA damages. Finally, we construct a polymer model that accounts for the local chromatin organization before and after a double-stranded DNA break (DSB) to estimate the level of chromatin decompaction. This novel passage time statistics method allows extracting transient dynamic at scales from one to few hundreds of nanometers, predicts the local changes in the number of binding molecules following DSB and can be used to better characterize the local dynamic of the chromatin.

biophysics↗