Biology subjects

Volk, T.

Publications and source records attributed to Volk, T..

9 recordsLinked to original sources

bioRxiv2026

Chromatin tethering to the nuclear envelope enhances its accessibility to RNAPII and promotes chromatin asymmetric organization

The intrinsic tendency of chromatin to self-attract competes with its association with RNA Polymerase II (RNAPII), a prerequisite for efficient transcription. Using high-resolution live imaging of chromatin and RNAPII organization in Drosophila larval muscle nuclei, we demonstrate that chromatin tethering to the nuclear envelope via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is essential for maintaining chromatin three-dimensional organization. Disruption of chromatin-lamina interactions either in LINC mutants or following knockdown of Barrier-to-Autointegration Factor (BAF) results in enhanced chromatin clustering in the nucleoplasm and reduced RNAPII-chromatin interaction. Consistent with these observations, computer simulations revealed an inverse relationship between the chromatin cluster size and the degree of chromatin tethering to the nuclear lamina. We also measured chromatin distribution with respect to the nuclear lamina and found asymmetry of RNAPII distributions within chromatin clusters, which correlated with their proximity to the nuclear envelope, a relationship that is lost in nuclei lacking a functional LINC complex. Our findings demonstrate that chromatin association with the nuclear envelope counteracts chromatin self-attraction and facilitates RNAPII binding to DNA.

molecular biology↗

bioRxiv2025

PKC Promotes T-Tubule Membrane Loss by Activating a PKD-NFκB Endocytic Pathway

BackgroundIn heart disease, the membrane of the cardiomyocyte transverse-axial tubular system (TATS) deteriorates. This impairs contractility, hinders recovery and predisposes to arrhythmia. However, the key signals and cellular processes driving TATS loss are not understood. We investigated protein kinase C (PKC) and its downstream signals in animal and human cardiomyocytes. MethodsVentricular cardiomyocytes were isolated from healthy adult rat, rabbit and failing human hearts and treated with the PKC activator phorbol 12-myristat 13-acetat (PMA) or receptor-mediated agonists, alongside inhibitors targeting PKC, PKD, NF{kappa}B, MKK1-ERK1/2, NFAT, or endocytic pathways. TATS density was analyzed by confocal microscopy using lipophilic membrane dyes. Signaling pathway activation was determined by Western blotting and RNA sequencing. Ca2+ signals and contractility were assessed in rat cells. Mechanisms of TATS loss were studied using endocytosis assays involving fixable dextran. ResultsPMA induced severe TATS loss, which was prevented by inhibiting PKC, PKD, NF{kappa}B or MKK1, but not by blocking NFAT or p38 MAPK. Receptor-mediated PKC activation also decreased TATS density. All effective inhibitors suppressed I{kappa}B expression. RNA sequencing indicated PMA-mediated activation of the NF{kappa}B and MAPK/ERK pathways and genes related to endocytosis. NF{kappa}B inhibition did not suppress the MAPK pathway, but MKK inhibition suppressed NF{kappa}B. PMA decreased Ca2+ transient amplitudes and contractility, whereas NF{kappa}B inhibitors preserved both. Dextran assays revealed that TATS membranes were internalized via a macropinocytic process that followed saturation kinetics was upregulated by PMA, downregulated by NF{kappa}B inhibition, and required PI3K, myosin I, and clathrin-independent endocytosis and correlated with the rate of TATS loss. Key findings were consistent in human cardiomyocytes and in ex-vivo rat and rabbit myocardial slice culture. ConclusionsPKC activation drives TATS loss in human and animal myocytes via PKC-PKD-NF{kappa}B, T-tubules are degraded by endocytic internalization, offering a new perspective on how cardiomyocyte membranes may deteriorate in heart disease.

cell biology↗

bioRxiv2023

Sedentary behavior may accelerate aging through impact on epigenome and transcriptome: lessons from muscle inactivation in Drosophila larvae

AimThe biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. Our animal model mimics sedentary behavior during early life, enabling us to explore the associated chromatin epigenetic and transcriptomic landscapes. MethodsDrosophila larvae carrying a temperature-sensitive mutation in the shibire1 (shi) gene were used. shi homozygous mutant larvae undergo instant arrest of muscle contraction at a restrictive temperature (30{degrees}C), without affecting other systems. Both shi and control (y,w) larvae were held at permissive temperature (18{degrees}C), and transferred to restrictive temperature (30{degrees}C) for six hours. Larvae were then dissected, fixed, and double-labeled with antibodies specific for epigenetic marks of chromatin activation (H3K9ac) and repression (H3K27me3), and their fluorescence signal was quantified. In addition, whole genome analysis of RNA-Pol II binding to DNA in muscle-specific inactive, or control larvae was performed using muscle-specific targeted DamID (TaDa) protocol. ResultsInducing muscle inactivity in shi larvae at 300C yielded a significantly higher ratio between chromatin activation and repression, based upon H3K9ac/H3K27me3 signals (p=0.025), relative to all control groups for which this ratio was comparable (p=0.995). Furthermore, muscle inactivation led to altered Pol II binding to 121 out of 2010 genes (6%). The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. In addition, a three-fold enrichment of genes coding for lncRNAs was noted in the muscle-inactive larvae. ConclusionInducing muscle inactivation exerted a multi-level impact upon chromatin, triggering an altered epigenetic balance, as well as downregulation of the transcriptional activity of genes essential for muscle function, carbohydrate metabolism, longevity and others. Extrapolating these findings to humans holds promise for establishing a molecular link between sedentary behavior and metabolic diseases.

physiology↗

bioRxiv2022

Regulation of chromatin microphase separation by adsorbed protein complexes

We show evidence of the association of RNA Polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g. RNAP). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn, modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

biophysics↗

bioRxiv2022

Functional LINC complex maintains epigenetic landscape and decondenses DNA in mature muscles

We show here that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is needed to minimize chromatin repression. The genomic binding profile of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb, and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications, and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

cell biology↗

bioRxiv2020

Mesoscale phase separation of chromatin in the nucleus

Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the compositional profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.

biophysics↗

bioRxiv2020

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Packaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin self-attractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior. This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

cell biology↗

bioRxiv2019

Mechanosensitive recruitment of BAF to the nuclear membrane inhibits nuclear E2F1 and Yap levels

Mechanotransduction has been implicated as an important factor in regulating cell cycle progression; however, the underlying mechanism has not been fully elucidated. Here, we describe a novel mechano-sensitive component, namely barrier to autointegration factor, (BAF), which regulates DNA endocycling in Drosophila muscle fibers. We show that BAF negatively regulates DNA endoreplication by inhibiting of the nuclear entrance of E2F1 and Yap/Yorkie, two key components in cell cycle control. Furthermore, BAF localization at the nuclear membrane is mechanosensitive, as it was downregulated in LINC mutant larval muscles, or following nuclear deformation caused by disruption of nucleus-sarcomere connections. BAF forms a protein complex with E2F1, which is sensitive to BAF phosphorylation. Knockdown of BAF kinase VRK1/Ball disrupted localization of BAF at the nuclear membrane and resulted in increased E2F1 nuclear levels. Taken together, our results reveal a novel mechanosensitive pathway controlling BAF phosphorylation and localization at the nuclear membrane, which in turn, represses nuclear accumulation of positive cell cycle regulators.

cell biology↗

bioRxiv2019

In vivo imaging of myonuclei during spontaneous muscle contraction reveals non-uniform nuclear mechanical dynamics in Nesprin/klar mutants

Muscle contractions produce reiterated cytoplasmic mechanical variations, which impact the nuclear membrane and potentially influence nuclear mechanotransduction. It is unclear, however, whether the mechanical dynamics of individual myonuclei changes during each contractile wave, and whether mutants whose connection to the cytoskeleton is impaired are subjected to different mechanical input.\n\nTo monitor nuclear mechanical dynamics in vivo, we imaged myonuclei along muscles during multiple spontaneous muscle contractile events, within live and intact Drosophila wild-type or Nesprin/klar mutant larvae. The data were subsequently analyzed and quantified aiming to reveal potential changes in the mechanical parameters of nuclear dynamics during muscle contraction in the Nesprin/klar mutant. Our results show that all myonuclei in control larvae exhibited comparable dynamics in the course of multiple contractile events. In contrast, myonuclei of homozygous mutant larvae lacking the Nesprin-like gene klar displayed differential dynamics relative to wild type, and higher variability between myonuclei at distinct positions along individual myofibers. Estimation of the drag force applied on individual myonuclei revealed that force fluctuations in time were considerably higher in the Nesprin/klar mutant myonuclei relative to control, reflecting a significant variability in the mechanical dynamics of individual myonuclei during contractile waves. The variable mechanical dynamics along the muscle fiber and the higher variance of Nesprin/klar mutant myonuclei may lead to altered nuclear mechanotransduction. Since mutations in Nesprin genes lead to devastating muscle and cardiac human diseases, our findings may provide new insight into the mechanism underlying these pathologies.

biophysics↗