Biology subjects

Engl, W.

Publications and source records attributed to Engl, W..

5 recordsLinked to original sources

bioRxiv2026

Senescence-induced Spatio-temporal Re-organization of the Nuclear Lamina Dynamic Landscape at Single-molecule Level in Live Human RPE Cells

A defining hallmark of cellular senescence is the extensive remodeling of the nuclear lamina (NL) in both structure, composition, and its interactions with lamina-associated genomic domains. However, how such senescence-induced changes could quantitatively impact the spatio-temporal dynamics of NL interactions at the single-molecule level remain poorly understood. Herein, using human retinal pigment epithelium (RPE) cells as model system, we establish a robust, chemically inducible cellular senescence model capable of recapitulating key signatures of the senescence-associated phenotype. Senescent RPE cells exhibit distinct alteration patterns in the expression of various NL components, including lamin B receptor (LBR) and emerin, two key mediators of NL-chromatin interactions. Importantly, live-cell diffusion measurements via imaging FCS reveal globally enhanced mobility for both LBR and emerin, indicative of weakened NL anchoring in senescent cells. Single-molecule tracking and super-resolved STAR mapping further unveil distinct, multi-modal re-organization of their NL-binding dynamics in both space and time (especially in the form of LBR binding "hotspots" where multiple stable binding events cluster to effect more extended and prolonged interaction), in line with their respective expression-level alterations and differential lamin interactions. Moreover, correlative mapping uncovers a strong spatial coupling between reduced LBR mobility and highly condensed constitutive heterochromatin domains at the NL, which become dispersed during senescence. Collectively, these findings demonstrate the re-organization of NL dynamic landscape at single-molecule level as a critical and previously under-explored facet of senescence-induced NL remodeling, and point to a synergistic interplay between physical de-anchoring, dynamic attenuation of stable NL-binding and spatial restructuring of chromatin architecture in modulating NL-heterochromatin contacts to potentially effect global changes in senescence-associated gene expression.

biophysics↗

bioRxiv2026

Nucleolar Condensation Orchestrates rRNA-dependent Mobility and Spatiotemporally Enriches rDNA-binding of Human Chromatin Remodeler BRG1

As key regulators of genome access via nucleosome translocation/ejection, SWI/SNF chromatin remodeling complexes share a core ATPase/translocase subunit, BRG1, central to their activity. Despite recent discovery of spatially clustered intranuclear "hotspots" where various SWI/SNF remodelers preferentially bind, the mechanistic driving force underlying such heterogeneous organization remains unclear. Herein, we show that human BRG1 undergoes condensation in vitro and in live cell nucleus, mediated by its IDR-rich C-terminus (BRG1C). Intranuclear condensates of BRG1C form across a wide range of (including endogenous) expression levels, are highly dynamic, and selectively partition into the fibrillar center of nucleolus, with their formation, localization and liquid-like properties governed primarily by patterned charge blocks in its sequence. Importantly, correlative single-molecule tracking and condensates mapping reveal rRNA-modulated constrained mobility and spatiotemporally enriched chromatin-binding to rDNA for BRG1C specifically within nucleolar condensates. These findings unveil a condensation-mediated coupling between remodeler dynamics and nucleolar architecture, pointing to a potentially generic mechanism for organizing remodeling activity in both space and time.

biophysics↗

bioRxiv2024

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2is FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

molecular biology↗

bioRxiv2023

Single-molecule Imaging of SWI/SNF Chromatin Remodelers Reveal Multi-modal and Cancer-mutant-specific Landscape of DNA-binding Dynamics

Chromatin remodeling, carried out by multi-subunit remodeler complexes, alleviates topological constraints posed by nucleosomes to regulate genome access. Although mutations in the SWI/SNF subfamily of remodelers are implicated in >20% of human cancers, how misregulation of their intranuclear dynamics could underpin cancer remains poorly understood. Combining single-molecule tracking and fluorescence correlation spectroscopy, we probed the live-cell dynamics of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), revealing temporally distinct modes characteristic of free and chromatin-associated diffusion and chromatin-binding. Quantifying residence times of the fully assembled remodeler complex further resolved one transient and two stable binding fractions. Moreover, super-resolved density mapping of single-molecule binding using a newly devised strategy, termed STAR, revealed heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events preferentially cluster, with particular enrichment of consecutive longer-lived stable binding. Importantly, we showed that the bromodomain plays a key role in modulating the enhancement of remodeler binding dynamics in a DNA-accessibility-dependent manner, but does not facilitate targeting to hyperacetylated chromatin. Finally, we compared the chromatin-binding dynamics of seven common BRG1 mutants implicated in various cancers across tumor types, and uncovered systematic alterations in residence time, binding frequency, fraction of time bound, targeting efficiency and number of binding "hotspots" unique to each point/truncation mutant. Collectively, our findings shed critical insight into the multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers to selectively modulate genome accessibility, and could potentially serve as quantitative, mutant-specific signatures for cancers associated with remodeling misregulation. SIGNIFICANCE STATEMENTUsing two complementary approaches, we performed, to our knowledge, the first single-molecule quantification of live-cell dynamics of the fully assembled human SWI/SNF remodeler complex by correlating three key common subunits, and uncovered distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner. Our super-resolved mapping of chromatin-binding also revealed intranuclear "hotspots" where remodelers bind repeatedly in nanometer-scale clusters, as a potential strategy to promote remodeling at these loci. By leveraging previously under-explored parameters, our findings revealed a broader and multi-modal landscape that regulates SWI/SNF-mediated remodeling dynamics in space and time, and established the biophysical basis for aberrant remodeler-chromatin interactions associated with seven mutants implicated in various cancers, which could potentially serve as their unique identifying yardsticks.

biophysics↗

bioRxiv2021

Cooperative regulation of adherens junction elongation through Epidermal Growth Factor Receptor (EGFR) activation.

The mechanisms controlling the elongation rates of adherens junctions are poorly characterized and lag behind our understanding of those controlling their static properties. In suspended cell aggregates, we found that the speed of de novo junction formation between two cells increases with the number of junctions that the cells are already engaged in. This cooperative effect is driven by the transient activation of the Epidermal Growth Factor Receptor (EGFR) upon junction formation. EGFR activation then regulates the actin cytoskeleton turnover while cortical tension remains unaltered. Overall, we show that EGFR activation regulates the elongation speed of junctions (kinetype) without affecting their final size (phenotype) in such aggregates.

biophysics↗