Biology subjects

Erpenbeck, L.

Publications and source records attributed to Erpenbeck, L..

8 recordsLinked to original sources

bioRxiv2026

Nanoscale Organization of Membrane Tension during Neutrophil Extracellular Trap Formation Revealed by Fluorescence Lifetime Imaging

Cells continuously generate and respond to mechanical forces across compartments, with the plasma membrane acting as a nanoscale interface for sensing and transmitting tension. How intracellular forces are translated into membrane tension during dynamic processes such as neutrophil extracellular trap (NET) formation remains unclear. Here, we combine the mechanosensitive fluorescent probe Flipper-TR with fluorescence lifetime imaging microscopy (FLIM) to map spatiotemporal changes in plasma membrane tension in living cells. After validation in HeLa and dHL-60 cells under osmotic perturbation, we apply this approach to primary human neutrophils undergoing NETosis. Membrane tension transiently increases during chromatin decondensation and nuclear swelling within 90 min and is followed by a marked decrease 30 min after membrane rupture. Prior to rupture, tension is spatially heterogeneous, indicating localized nanoscale mechanical regulation. Cholesterol depletion abolishes the transient increase and reduces heterogeneity without affecting overall NETosis kinetics. Together, these findings establish the plasma membrane as a dynamic nanoscale reporter of intracellular mechanical stress during NETosis.

immunology↗

bioRxiv2026

Simulation of neurotransmitter release and its imaging by fluorescent sensors

Cells release signaling molecules such as neurotransmitters that diffuse through the extracellular space and bind to receptors. These signaling molecules can be detected by fluorescent sensors/probes to provide images of the signaling process. Such images are not equivalent to a concentration because diffusion and sensor kinetics affect (convolute) them. Therefore, computational approaches are necessary to disentangle these contributions and allow interpretation of fluorescent sensor-based images. Here, we present a kinetic Monte Carlo framework (FLuorescence Imaging Kinetic Simulation, FLIKS) that simulates signaling molecules undergoing cellular release, stochastic diffusion and reversible binding to sensors in realistic cellular (2D or 3D) geometries. We apply it to model neurotransmitter (dopamine) release in synaptic clefts and for paracrine signaling by immune cells. We also show how sensor location, sensor kinetics and release location affect fluorescence images. For example, we show how sensor sensitivity depends on the distance from the synaptic cleft and changes when dopamine transporters (DAT) clear dopamine. The approach also allows to compare the performance of membrane bound (genetically encoded) sensors versus artificial sensors such as nanosensors placed outside under or around the cells. As an example, we also demonstrate how the images of catecholamine release by immune cells can be modeled and compared to experimental data to better understand the release pattern. This framework provides a quantitative basis for analyzing and interpreting fluorescent sensor imaging data.

neuroscience↗

bioRxiv2025

Rapid catecholamine trafficking regulates neutrophil functions and neutrophil-platelet interactions

Neutrophils are key effector cells of the innate immune system that respond to small signaling molecules regulating immune responses. For a long time, similarities between neuronal and immune cells have been discussed. Here, we show that human neutrophils rapidly take up, package and use catecholamine neurotransmitters such as dopamine or epinephrine via the machinery known from neurons. Uptake and release of catecholamines as well as trafficking and packaging into MPO/VMAT2-positive primary vesicles is visualized with false fluorescent neurotransmitters. We also directly image the fast (> 10 s) and transient release of catecholamines from neutrophils with near infrared fluorescent nanosensors. Serotonin or activated platelets trigger calcium (Ca2+) signaling and consequently exocytosis of catecholamines. They reduce NET-formation but increase platelet aggregation. Thus, we establish similarities between neurons and neutrophils and identify a paracrine neutrophil-platelet feedback loop relevant for inflammatory and coagulatory conditions.

immunology↗

bioRxiv2024

Primary Human Neutrophils and Monocytes/Macrophages Migrate along Endothelial Cell Boundaries to Optimize Search Efficiency

Neutrophils and monocytes/macrophages are sentinels of inflammatory signals. To reach the sites of action, both cell types attach to and then transmigrate the endothelial cell layer that lines the luminal side of blood vessels. While it has been reported that neutrophils and monocytes/macrophages actively migrate along the surface of the vasculature, it remains elusive if and how these motion pattern augment the efficiency of the immune system. Here, we conducted co-culture experiments of primary human monocytes and neutrophils, respectively, with human umbilical vein endothelial cells (HUVECs). Combining classical biomedical approaches with quantitative image analysis and numerical models, we find that immune cells simultaneously increase the number of sampled cells vs. traveled distance and sensitivity to chemokines by migrating along endothelial cell-cell boundaries. Collectively, these findings establish search optimization of neutrophils and monocytes/macrophages through limitation of motion pattern to cell-cell boundaries.

cell biology↗

bioRxiv2022

Expansion microscopy of nuclear structure and dynamics in neutrophils

Neutrophils are key players of the immune system and possess an arsenal of effector functions, including the ability to form and expel neutrophil extracellular traps (NETs) in a process termed NETosis. During NETosis, the nuclear DNA/chromatin expands until it fills the whole cell and is released into the extracellular space. NETs are composed of DNA decorated with histones, proteins or peptides and NETosis is implicated in many diseases. Resolving the structure and dynamics of the nucleus in great detail is essential to understand the underlying processes but so far super-resolution methods have not been applied. Here, we developed an expansion microscopy-based method and determined the spatial distribution of chromatin/DNA, histone H1, and nucleophosmin (NPM1) with a 4.9-fold improved resolution (< 40 nm) and increased information content. It allowed us to identify the punctate localization of NPM1 in the nucleus and histone-rich domains in NETotic cells with a size of 54 nm. The technique could also be applied to components of the nuclear envelope (lamins B1 and B2) and myeloperoxidase (MPO) providing a complete picture of nuclear dynamics and structure. In conclusion, expansion microscopy enables super-resolved imaging of the highly dynamic structure of nuclei in immune cells. Why it mattersAccessibility to high-resolution imaging is critical to advancing research across various disciplines. However, conventionally this requires demanding optical hardware, special fluorophores or data analysis. Expansion microscopy is a technique adaptable to different cell and tissue types and is comparatively inexpensive and easy to perform. Applying this technique to cells and compartments such as the nucleus of immune cells that are difficult to image due to their size and morphology, yields valuable structural insights that would otherwise require more difficult super-resolution methods.

biophysics↗

bioRxiv2019

Programmed transport and release of nanoscale cargo by immune cells

Transport and delivery of (nanoscale) materials are crucial for many applications in biomedicine. However, controlled uptake, transport and triggered release of such cargo remains challenging. In this study, we use human immune cells (neutrophilic granulocytes, neutrophils) and program them to perform these tasks in vitro. For this purpose, we let neutrophils phagocytose a nanoscale cargo. As an example, we used DNA-functionalized single-walled carbon nanotubes (SWCNT) that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling and subsequent rupture of the cellular membrane and release of the cells whole content. By using the process of NETosis we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation with LPS (100 g/ml), cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release their cargo. The transported and released SWCNT sensor cargo is still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils can be used for programmed transport and delivery of functional nanomaterials.

biophysics↗

bioRxiv2019

Exfoliated near infrared fluorescent CaCuSi4O10 nanosheets with ultra-high photostability and brightness for biological imaging

Imaging of complex (biological) samples in the near infrared (nIR) range of the spectrum is beneficial due to reduced light scattering, absorption, phototoxicity and autofluorescence. However, there are only few near infrared fluorescent materials known and suitable for biomedical applications. Here, we exfoliate the layered pigment CaCuSi4O10 (known as Egyptian Blue, EB) via facile tip sonication into nanosheets (EB-NS) with ultra-high nIR fluorescence stability and brightness. The size of EB-NS can be tailored by tip sonication to diameters < 20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the total fluorescence intensity scales with the number of Cu2+ ions that serve as luminescent centers. Furthermore, EB-NS display no bleaching and ultra-high brightness compared to other nIR fluorophores. The versatility of EB-NS is demonstrated by in vivo single-particle tracking and microrheology measurements in developing Drosophila embryos. Additionally, we show that EB-NS can be uptaken by plants and remotely detected in a low cost stand-off detection setup despite strong plant background fluorescence. In summary, EB-NS are a highly versatile, bright, photostable and biocompatible nIR fluorescent material that has the potential for a wide range of bioimaging applications both in animal and plant systems.

biophysics↗

bioRxiv2019

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Youngs modulus E) within the physiologically relevant range from 1 kPa to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

biophysics↗