Biology subjects

Houtsmuller, A. B.

Publications and source records attributed to Houtsmuller, A. B..

8 recordsLinked to original sources

bioRxiv2023

A minimum threshold for myelination of pyramidal cells in human and mouse neocortex

Neocortical pyramidal neurons are frequently myelinated. Diversity in the topography of axonal myelination in the cerebral cortex has been attributed to a combination of electrophysiological activity, axonal morphology, and neuronal-glial interactions. Previously, we showed that axonal segment length and calibre are critical determinants of fast-spiking interneuron myelination (Stedehouder, J. et al (2019). However, the factors that determine the myelination of individual axonal segments along neocortical pyramidal neurons remain largely unexplored. Here, we used structured illumination microscopy and cell type-specific manipulations to examine the extent to which axonal morphology determines the topography of axonal myelination in mouse neocortical pyramidal neurons. We found that, unlike what was determined for fast-spiking interneurons, the joint combination of axonal calibre and interbranch distance does not predict axonal myelination in pyramidal neurons, rather it provides a minimum threshold for myelination; pyramidal neurons with an axon calibre and interbranch distance lower than 0.24 {micro}m and 19 {micro}m, respectively, are almost never myelinated. Moreover, we further confirmed that these findings in mice also extend to human neocortical pyramidal cell myelination, suggesting that this mechanism is evolutionarily conserved. Taken together, our findings suggest that axonal morphology is highly deterministic of the topography and cell-type specificity of neocortical myelination.

neuroscience↗

bioRxiv2022

PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

How steroid hormone receptors (SHRs) orchestrate transcriptional activity remains only partly understood. Upon activation, SHRs bind the genome and recruit their co-regulators, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we comprehensively dissected the Glucocorticoid Receptor (GR) co-regulatory complex involved in gene-target regulation. We describe a novel functional cross-talk between PAXIP1 and the cohesin subunit STAG2 that is critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on the genome, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. Moreover, in lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

genomics↗

bioRxiv2022

Correlative light and electron microscopy reveals fork-shaped structures at actin entry sites of focal adhesions

Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

cell biology↗

bioRxiv2022

Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy to study how the two recombinases are organized on the ssDNA at individual DSBs, using a mouse model with severe defects in meiotic DSB formation and synapsis: Hormad1-/-. In accordance with previous analyses, we most frequently observed recombinase configurations with 1 DMC1 and 1 RAD51 nanofocus (D1R1), or two DMC1 nanofoci combined with a single RAD51 nanofocus (D2R1), and established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. In wild type, the D1R1:D2R1 frequency ratio decreased over time, but in the knockout, this ratio did not change due to increased D2R1 and decreased D1R1 foci frequency in leptotene. Also, the Hormad1-/- nanofoci were smaller. Nearest neighbor analysis of RAD51/DMC1 nanofoci showed two preferred distances at [~]300 and [~]900 nm in wild type, but only at [~]300 nm in Hormad1-/-. Combined with the lower total number of DSBs in the mutant, this suggests that a preferred distance of 900 nm represents the distance between DSB sites. We propose models with the D1R1 configuration representing a scenario with only one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or with both ends loaded by the two recombinases, but in below-resolution proximity. Surprisingly, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting the formation of coils in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. In conclusion, our data show that HORMAD1 affects the lifetime of recombinase-accumulation patterns at meiotic DSBs, as well as the structure of the synaptonemal complex. Author summaryMeiosis is a specialized cell division which generates haploid germ cells. In order to correctly pair homologous chromosomes in the first meiotic prophase, repair of programmed double strand breaks (DSBs) is essential. By unravelling molecular details of the protein assemblies at single DSBs, using super-resolution microscopy, we aim to understand the dynamics of repair intermediates and their functions. To this end, we investigated the localization of the two recombinases RAD51 and DMC1 in wild type and HORMAD1-deficient cells. HORMAD1 is involved in multiple aspects of homologous chromosome association: it regulates formation and repair of DSBs, and it stimulates formation of the synaptonemal complex, the macromolecular protein assembly that connects paired chromosomes. RAD51 and DMC1 enable chromosome pairing by promoting the invasions of the intact chromatids by single-stranded DNA ends that result from DSBs. We found that, in the absence of HORMAD1, RAD51 and DMC1 showed small but significant morphological and positional changes, combined with altered kinetics of specific RAD51/DMC1 configurations. We also observed changes in the structure of the synaptonemal complex in Hormad1-/ spermatocytes. This study contributes to a better understanding of the molecular details of meiotic homologous recombination and the role of HORMAD1 in meiotic prophase.

developmental biology↗

bioRxiv2020

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces drastic chromosome condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to reestablish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation, super-resolution imaging and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon reentry into G1. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

genomics↗

bioRxiv2020

EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

Extracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

bioengineering↗

bioRxiv2019

Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A and RAD21, cohesin contains in somatic cells one of two orthologous STAG subunits, SA1 or SA2. How these variable subunits affect the function of the cohesin complex is still unclear. SA1- and SA2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of SA1 and SA2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either SA1 or SA2. Following rapid depletion of either subunit, we perform high resolution Hi-C, RNA-sequencing and sequential ChIP studies to show that SA1 and SA2 do not co-occupy individual binding sites and have distinct ways how they affect looping and gene expression. These findings are supported at the single cell level by single-molecule localizations via dSTORM super-resolution imaging. Since somatic and congenital mutations of the SA subunits are associated with cancer (SA2) and intellectual disability syndromes with congenital abnormalities (SA1 and SA2), we verified SA1-/SA2-dependencies using human neural stem cells, hence highlighting their importance for understanding particular disease contexts.

cell biology↗

bioRxiv2019

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.

cell biology↗