Márton Gelléri

Double-strand breaks (DSBs) are critical lesions in genomic DNA, and their accurate repair is essential for maintaining genome stability. The nuclear actin cytoskeleton has been implicated in homology-directed repair (HDR) of DSBs. However, the underlying mechanism remains poorly understood. Here, we report that Myosin VI (Myo6), an actin-based motor protein, cooperates with F-actin in end resection and DSB mobilization. Our findings reveal that Myo6 directly interacts with both KU70 and the ubiquitin-dependent segregase VCP to facilitate the extraction of the KU70/80 complex from chromatin. This process is supported by F-actin, revealing an interplay between nuclear actin dynamics and the DSB repair machinery. By elucidating the function of Myo6 and its direct interactions with key repair factors, our study provides mechanistic insight into how repair mechanisms rely on nuclear actin to safeguard genome integrity.

Transcriptional condensates at super-enhancers are thought to concentrate BRD4, Mediator, and RNA polymerase II (Pol II) to promote gene activation, yet their compositional organization and regulation remain poorly understood. We developed a high-throughput live-cell phenomics platform based on endogenous fluorescent tagging of BRD4, MED14 (Mediator), and POLR2A (Pol II) to systematically quantify transcriptional condensate states across >1,000 chemical perturbations. Contrary to prevailing models of largely co-occupied assemblies, we find compositionally heterogenous condensate populations. In particular, BRD4-only spots emerged as a prominent class that is depleted of Mediator and Pol II, enriched at chromatin, and resistant to transcription initiation inhibition. Mechanistically, compound screening coupled to mechanism-of-action analysis identifies histone acetylation as a dominant regulatory axis for BRD4-only spots: Bromodomain and Extra-Terminal motif (BET) and histone acetyltransferase inhibition selectively deplete BRD4-only condensates, while histone deacetylase inhibition expands them. Together, these findings support a model in which acetylation-dependent BRD4 condensates define a distinct chromatin-associated regulatory state that is separable from canonical transcriptionally engaged condensates. More broadly, our work establishes condensate composition as a quantitative phenotype and provides a scalable framework for systematically dissecting the regulation of condensates across perturbations, cell types, and disease contexts.

SUMO, the small ubiquitin-like modifier, modulates interactions of folded proteins, but also affects the dynamics of biomolecular condensates. Moreover, SUMO can directly impinge on the biophysical properties of its targets, often increasing their solubility. TDP-43 is an RNA-binding protein containing an intrinsically disordered domain that participates in protein phase separation. Its aggregation, linked to neurodegenerative diseases, is counteracted by SUMOylation, but the underlying principles are poorly understood. We now reveal distinct mechanisms of how the two isoforms, SUMO1 and SUMO2, act on TDP-43. While SUMO1 inhibits phase separation by blocking TDP-43’s self-assembly site, SUMO2 enhances phase separation via proximity-induced self-interaction involving its N-terminus and promotes a liquid-like state that prevents aggregation. The latter represents a general principle by which SUMO solubilises other targets. Our findings provide insight into how SUMOylation regulates transitions between soluble, liquid-like and aggregated states.

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly found at the interface between chromatin and the interchromatin space called euchromatin. At present, the characterization of eu- and heterochromatin is mostly based on epigenetic modifications of histone proteins along the DNA sequence, while little is known about absolute DNA densities across the cell nucleus and their functional implications. Models of the nucleus solely based on biochemical data and assumptions about the nature of chromatin as a polymer still differ fundamentally from imaging data generated by high-resolution microscopy. This indicates that there is still important structurally relevant information missing. We believe that spatial constraints might be involved in gene regulation and have therefore developed a method that allows the measurement of absolute DNA densities in mammalian cell nuclei by transforming super-resolution localization data into true-to-scale density maps by Voronoi tessellation.

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly found at the interface between chromatin and the interchromatin space called euchromatin. At present, the characterization of eu- and heterochromatin is mostly based on epigenetic modifications of histone proteins along the DNA sequence, while little is known about absolute DNA densities across the cell nucleus and their functional implications. Models of the nucleus solely based on biochemical data and assumptions about the nature of chromatin as a polymer still differ fundamentally from imaging data generated by high-resolution microscopy. This indicates that there is still important structurally relevant information missing. We believe that spatial constraints might be involved in gene regulation and have therefore developed a method that allows the measurement of absolute DNA densities in mammalian cell nuclei by transforming super-resolution localization data into true-to-scale density maps by Voronoi tessellation.

Stimulated emission depletion (STED) microscopy, a key optical super-resolution imaging method, has extended our ability to view details to resolution levels of tens of nanometers. Its resolution depends on fluorophore de-excitation efficiency, and increases with depletion laser power. However, high-power irradiation permanently turns off the fluorescence due to photo-bleaching of the fluorophores. As a result, there is a trade-off between spatial resolution and imaging time. Here, we overcome this limitation by introducing reactivatable STED (ReSTED) based on the photophysical properties of the nanographene dibenzo[hi,st]ovalene (DBOV). In contrast to the photo-induced decomposition of other fluorophores, the fluorescence of DBOV is only temporarily deactivated and can be reactivated by near-infrared light (including the 775 nm depletion beam). As a result, this fluorophore allows for hours-long, high-resolution 3D STED imaging, greatly expanding the applications of STED microscopy.

The synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. Here we report three novel findings suggesting a completely new role of SV2A. First, we demonstrate that SV2A is localized at the outer mitochondrial membrane (OMM) using confocal and super-resolution microscopy. Second, Inactivation of SV2A in our cell and animal models leads to fragmented mitochondria. In addition, SV2A also affects the basal autophagic flux as well as mitophagy. Third, using proteomics analysis we demonstrate that SV2A interacts with the fission factor DRP1 and the autophagy factor ATG9A. Using AlphaFold3 we provide a first glimpse of the molecular interaction between DRP1 and SV2A. Our findings demonstrate that SV2A is not only a vesicular protein but also a mitochondrial protein in the OMM with defined functions regulating mitochondrial morphology and autophagy.

The synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. Here we report three novel findings suggesting a completely new role of SV2A. First, we demonstrate that SV2A is localized at the outer mitochondrial membrane (OMM) using confocal and super-resolution microscopy. Second, Inactivation of SV2A in our cell and animal models leads to fragmented mitochondria. In addition, SV2A also affects the basal autophagic flux as well as mitophagy. Third, using proteomics analysis we demonstrate that SV2A interacts with the fission factor DRP1 and the autophagy factor ATG9A. Using AlphaFold3 we provide a first glimpse of the molecular interaction between DRP1 and SV2A. Our findings demonstrate that SV2A is not only a vesicular protein but also a mitochondrial protein in the OMM with defined functions regulating mitochondrial morphology and autophagy.

Thioflavin T (ThT) is a typical dye used to visualize the aggregation and formation of fibrillar structures, e.g., amyloid fibrils and peptide nanofibrils. ThT has been considered to produce stable fluorescence when interacting with aggregated proteins. For single-molecule localization microscopy (SMLM)-based optical super-resolution imaging, a photoswitching/blinking fluorescence property is required. Here we demonstrate that, in contrast to previous reports, ThT exhibits intrinsic stochastic blinking properties, without the need for blinking imaging buffer, in stable binding conditions. The blinking properties (photon number, blinking time, and on-off duty cycle) of ThT at the single-molecule level (for ultralow concentrations) were investigated under different conditions. As a proof of concept, we performed SMLM imaging of ThT-labeled α-synuclein fibrils measured in air and PBS buffer.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.