Biology subjects

Joosten, B.

Publications and source records attributed to Joosten, B..

5 recordsLinked to original sources

bioRxiv2026

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

biophysics↗

bioRxiv2021

IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

Actin plays a central role in many cell biological processes including division and motility. Mammals have six, highly conserved actin isoforms with nonredundant biological functions, yet the molecular basis of isoform specificity remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoform function in fixed and living cells. We first identified a residue pair in {beta}-actin that permits non-disruptive tag integration. Next, we used knock-in cell lines to demonstrate that the expression and filament incorporation of IntAct {beta}-actin is indistinguishable from wildtype. Furthermore, IntAct {beta}-actin remains associated with actin-binding proteins profilin, cofilin and formin family members DIAPH1 and FMNL2 and can be targeted in living cells. To demonstrate the usability of IntAct for actin isoform investigations, we also generated IntAct {gamma}-actin cells and show that actin isoform specific distribution remains unaltered in human cells. Moreover, introduction of tagged actin variants in yeast demonstrated an expected variant-dependent incorporation into patches and filaments. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions.

cell biology↗

bioRxiv2020

Characterization of the signalling modalities of prostaglandin E2 receptors EP2 and EP4 reveals crosstalk and a role for microtubules

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signalling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gs protein, EP4 also couples to the inhibitory Gi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signalling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signalling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signalling in myeloid cells. Considering that modulation of PGE2 signalling is regarded as an important therapeutic possibility in anti-tumour immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

immunology↗

bioRxiv2019

Intracellular Galectin-9 controls dendritic cell function by maintaining plasma membrane rigidity

Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.

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↗