Biology subjects

Schaaf, M.

Publications and source records attributed to Schaaf, M..

6 recordsLinked to original sources

bioRxiv2026

Inflammation reshapes epidermal and leukocyte differentiation resulting in altered glucocorticoid responsiveness

Glucocorticoids are widely prescribed anti-inflammatory drugs that act on nearly all cell types due to the ubiquitous expression of the glucocorticoid receptor (GR). While the molecular mechanisms of GR activation have been studied extensively, it is less well understood how GR signaling modulates cell type-specific responses during inflammation. Using the unique attributes of the zebrafish, we performed single-cell RNA sequencing to characterize GR-mediated transcriptional regulation under conditions of wounding-induced inflammation. We found specific cell populations that occurred only under inflammatory conditions within epidermal cells, macrophages and neutrophils. The inflammation-specific subset of epidermal cells showed elevated expression of inflammatory markers, and these cells exhibited a reduced transactivation response to dexamethasone. This reduction in GR activation was also observed in the inflammation-specific subset of macrophages, but not in neutrophils, which retained dexamethasone responsiveness. By correlating the dexamethasone responsiveness with transcriptional coregulator expression, we identified candidate regulatory mechanisms underlying differential GR responsiveness across cell types. Taken together, we provided unique mechanistic insights into cell differentiation during a local inflammatory response and cell type-specific glucocorticoid responsiveness in vivo.

immunology↗

bioRxiv2025

Functional Mapping and Engineering of the Sec Translocon Unlocked by a Cell-Free System

Almost all proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active variants. We further leverage our system to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.

molecular biology↗

bioRxiv2022

Multifocal two-photon excitation fluorescence microscopy reveals hop diffusion of H-Ras membrane anchors in epidermal cells of zebrafish embryos

Developments in fluorescence microscopy techniques have enabled imaging of individual fluorescently labelled proteins in biological systems, and in the current study, a single-molecule microscopy (SMM) technique has been applied in vivo, using the zebrafish embryo model. We have used multifocal two-photon excitation fluorescence microscopy (2PEFM) to study the dynamics of a GFP-fused H-Ras membrane-anchoring domain, GFP-C10H-Ras, in the epidermal cells of living embryos. In previous studies, a fast and a slow diffusing population of GFP-C10H-Ras molecules had been found. The application of the multifocal 2PEFM technique enabled us to focus on the slow diffusing population, which appears to occur in clusters that diffuse within microdomains of the epidermal cell membranes. Based on their mobility on a short timescale ([≤] 1s) we could distinguish between a subpopulation that was diffusing and one that was virtually immobile. Owing to the multifocal 2PEFM imaging mode, we were able to dramatically reduce photobleaching which enabled us to follow the GFP-C10H-Ras particles over a prolonged time (> 3 s) and reconstruct their molecular trajectories of the diffusing subpopulation. These trajectories exhibited that the C10H-Ras particles continuously switch between a diffusing state and brief bursts of increased diffusion. As a result, they display an anomalous mobility pattern that can be referred to as hop diffusion. Taken together, this study demonstrates that multifocal 2PEFM offers a powerful approach to studying individual particles for prolonged periods of time, and that using this approach we were able to uncover the hopping behavior of GFP-C10H-Ras. SUMMARY STATEMENTBy application of the two-photon excitation single-molecule microscopy to living zebrafish embryos, anomalous diffusion modes of individual H-Ras membrane anchors in epidermal cells were found.

biophysics↗

bioRxiv2021

Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

Over the last two decades, developments in single-molecule microscopy (SMM) have enabled imaging and tracking of individual, fluorescently labelled proteins in biological systems, and most of these studies have focused on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied the protein dynamics of the membrane protein H-Ras, mutants of this protein, and its membrane-anchoring domain, C10H-Ras, in epidermal cells of living two-day-old embryos, using a total internal reflection fluorescence microscopy (TIRFM) setup. For all proteins studied, our results consistently confirm the presence of a fast- and a slow-diffusing subpopulations of molecules, which both confine to microdomains within the plasma membrane. Although the mobility patterns of H-Ras, mutants of this proteins and its membrane-anchoring domain were remarkably similar, the constitutively active H-Ras mutant, H-RasV12, exhibited significantly higher diffusion rates than the wild-type H-Ras and its inactive mutant, H-RasN17. Ultimately, we characterized variability in our data obtained using the zebrafish embryo model and demonstrated that differences between cells within the same embryo are the largest source of variation in our data. Our findings are in line with a model in which the cellular architecture of individual cells within a tissue determine the mobility of H-Ras proteins anchored in the plasma membrane cytoplasmic leaflet. Thereby, our results underline the growing importance of performing SMM imaging in vivo in order to better understand factors influencing the protein dynamics in an intact living organism. SUMMARY STATEMENTBy application of single-molecule microscopy to living zebrafish embryos, factors altering the in vivo dynamics of H-Ras proteins in epidermal cells were analyzed.

biophysics↗

bioRxiv2020

Glucocorticoid treatment exacerbates mycobacterial infection by reducing the phagocytic capacity of macrophages

Glucocorticoids are effective drugs for treating immune-related diseases, but prolonged therapy is associated with an increased risk of various infectious diseases, including tuberculosis. In this study, we have used a larval zebrafish model for tuberculosis, based on Mycobacterium marinum (Mm) infection, to study the effect of glucocorticoids. Our results show that the synthetic glucocorticoid beclomethasone increases the bacterial burden and the dissemination of a systemic Mm infection. The exacerbated Mm infection was associated with a decreased phagocytic activity of macrophages, higher percentages of extracellular bacteria, and a reduced rate of infected cell death, whereas the bactericidal capacity of the macrophages was not affected. The inhibited phagocytic capacity of macrophages was associated with suppression of the transcription of genes involved in phagocytosis in these cells. The decreased bacterial phagocytosis by macrophages was not specific for Mm, since it was also observed upon infection with Salmonella Typhimurium. In conclusion, our results show that glucocorticoids inhibit the phagocytic activity of macrophages, which may increase the severity of bacterial infections like tuberculosis. Summary statementUsing a zebrafish tuberculosis model, we show that glucocorticoids decrease phagocytosis by macrophages, thereby increasing the bacterial burden. This may explain the glucocorticoid-induced increase in susceptibility to tuberculosis in humans.

immunology↗

bioRxiv2019

Early life glucocorticoid exposure modulates immune function in zebrafish (Danio rerio) larvae

In this study we have assessed the effects of increased cortisol levels during early embryonic development on immune function in zebrafish (Danio rerio) larvae. Fertilized eggs were exposed to either a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor selectively) or a control medium for 6 hours post-fertilisation (0-6 hpf). First, we measured baseline expression of a number of immune-related genes (socs3a, mpeg1.1, mpeg1.2 and irg1l) 5 days post-fertilisation (dpf) in larvae of the AB and TL strain to assess the effectiveness of our exposure procedure and potential strain differences. Cortisol and dexamethasone strongly up-regulated baseline expression of these genes independent of strain. The next series of experiments were therefore carried out in larvae of the AB strain only. We measured neutrophil/macrophage recruitment following tail fin amputation (performed at 3 dpf) and phenotypical changes as well as survival following LPS-induced sepsis (150 g/ml; 4-5 dpf). Dexamethasone, but not cortisol, exposure at 0-6 hpf enhanced neutrophil recruitment 4 hours post tail fin amputation. Cortisol and dexamethasone exposure at 0-6 hpf led to a milder phenotype (e.g. less tail fin damage) and enhanced survival following LPS challenge compared to control exposure. Gene-expression analysis showed accompanying differences in transcript abundance of tlr4bb, cxcr4a, myd88, il1{beta} and il10. These data show that early-life exposure to cortisol, which may be considered to be a model or proxy of maternal stress, induces an adaptive response to immune challenges, which seems mediated via the glucocorticoid receptor.

immunology↗