Biology subjects

Brouns, T.

Publications and source records attributed to Brouns, T..

5 recordsLinked to original sources

bioRxiv2024

HIV integrase compacts viral DNA into biphasic condensates

The human immunodeficiency virus (HIV) infects non-dividing cells and its genome must be compacted to enter the cell nucleus. Here, we show that the viral enzyme integrase (IN) compacts HIV DNA mimetics in vitro. Under physiological conditions, IN-compacted genomes are consistent in size with those found for pre-integration complexes in infected cells. Compaction occurs in two stages: first IN tetramers bridge DNA strands and assemble into "rosette" structures that consist of a nucleo-protein core and extruding bare DNA. In a second stage, the extruding DNA loops condense onto the rosette core to form a disordered and viscoelastic outer layer. Notably, the core complex is susceptible towards IN inhibitors, whereas the diffuse outer layer is not. Together, our data suggest that IN has a structural role in viral DNA compaction and raise the possibility to develop inhibitors that target IN-DNA interactions in disordered condensates. TeaserSingle-molecule studies demonstrate the mechanism, dynamics, and drug-susceptibility of viral genome compaction by HIV integrase.

biophysics↗

bioRxiv2023

High-yield, ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single molecule manipulation assays

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCR reactions: first [~]380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR reaction to generate [~]kbp long double-stranded DNA constructs with multiple labels at the respective ends. We use DBCO-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under force, with an average lifetime of 60 {+/-} 3 hours at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

biophysics↗

bioRxiv2020

High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced transient nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ~10,000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but unlike to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes, likely due to their shorter N-terminal -helix. Finally, our results reconcile previously conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability.

biophysics↗

bioRxiv2019

Assessing the utility of MAGNETO to control neuronal excitability in the somatosensory cortex

Magnetic neuromodulation has outstanding promise for the development of novel neural interfaces without direct physical intervention with the brain. Here we tested the utility of Magneto in the adult somatosensory cortex by performing whole-cell intracellular recordings in vitro and extracellular recordings in freely moving mice. Results show that magnetic stimulation does not alter subthreshold membrane excitability or contribute to the generation of action potentials in virally transduced neurons expressing Magneto.

neuroscience↗

bioRxiv2019

PASER for automated analysis of neural signals recorded in pulsating magnetic fields

Thanks to the advancements in multichannel intracranial neural recordings, magnetic neuroimaging and magnetic neurostimulation techniques (including magnetogenetics), it is now possible to perform large-scale high-throughput neural recordings while imaging or controlling neural activity in a magnetic field. Analysis of neural recordings performed in a switching magnetic field, however, is not a trivial task as gradient and pulse artefacts interfere with the unit isolation. Here we introduce a toolbox called PASER, Processing and Analysis Schemes for Extracellular Recordings, that performs automated denoising, artefact removal, quality control of electrical recordings, unit classification and visualization. PASER is written in MATLAB and modular by design. The current version integrates with third party applications to provide additional functionality, including data import, spike sorting and the analysis of local field potentials. After the description of the toolbox, we evaluate 9 different spike sorting algorithms based on computational cost, unit yield, unit quality and clustering reliability across varying conditions including self-blurring and noise-reversal. Implementation of the best performing spike sorting algorithm (KiloSort) in the default version of the PASER provides the end user with a fully automated pipeline for quantitative analysis of broadband extracellular signals. PASER can be integrated with any established pipeline that sample neural activity with intracranial electrodes. Unlike the existing algorithmic solutions, PASER provides an end-to-end solution for neural recordings made in switching magnetic fields independent from the number of electrodes and the duration of recordings, thus enables high-throughput analysis of neural activity in a wide range of electro-magnetic recording conditions.

neuroscience↗