Biology subjects

Ragaller, F.

Publications and source records attributed to Ragaller, F..

8 recordsLinked to original sources

bioRxiv2026

Cell surface remodeling caused by the loss of TMEM30A in immune cells

Plasma membrane lipid asymmetry is tightly regulated and fundamental to mammalian cell physiology. TMEM30A is the {beta}-subunit of P4-ATPases, flippase enzymes that maintain strict phosphatidylserine (PS) asymmetry by pumping it from the outer to the cytosolic leaflet. Loss of TMEM30A function causes constitutive PS externalization and has been implicated in diseases such as diffuse large B-cell lymphoma and tumour immune evasion. Here, we systematically define the biophysical and molecular consequences of TMEM30A deletion in immune cells. Using a live-cell lipid reporter, membrane order probe, and surface proteome mapping, we show that TMEM30A-knockout cells display robust PS externalization accompanied by faster lateral diffusion of membrane constituents and decreased plasma membrane order. Surface proteome reorganization includes increased abundance of tetraspanins and CD47. Further, TMEM30A loss triggers glycocalyx remodeling via ADAM10-dependent shedding that removes major transmembrane mucins, including CD43 and CD162. Together, these data reveal a coordinated reorganization of lipids, glycans, and proteins upon TMEM30A loss, suggesting mechanistic links between flippase dysfunction and increased plasma membrane dynamics and potential sensitization to immune therapy. Furthermore, our study provides an integrated surfaceome framework that might shed light on the relationship between TMEM30A expression and clinical outcomes in cancer.

immunology↗

bioRxiv2025

Studying macromolecular composition in cell-cell interfaces using 3D membrane reconstitution systems

During direct communication between two cells, the plasma membranes of each cell serve as a platform for ligand-receptor interaction initiating downstream signalling cascades. In immune cell signalling, this cell-cell interface - the immune synapse - is highly spatiotemporally organized. Multiple stimulatory and co-stimulatory signals need to be integrated over time to ensure proper immune cell function. This process is still not fully understood given the vast complexity of interactions between proteins, lipids, glycocalyx and associated cortical actin cytoskeleton. To examine the impact of a single component, the use of model membrane systems has increased. Here, we developed a fully artificial system to study the interface between two vesicles and a semi-artificial one between a live cell and a vesicle to reconstitute 3D contacts. We investigated the distribution and reorganization of immune cell proteins at artificial and semi-artificial contacts. Using our vesicle-vesicle system, we show the enrichment and depletion of different proteins in the synapse. Using the cell-vesicle system we showed how different peptides with varying affinity presented by the same MHC class I affect T cell activation. We further explored the distribution of glycocalyx elements at the cell-cell contact and showed differential partitioning of different sugar moieties in the interface. While we focused on the T cell interface here, our model systems are powerful tools to study distribution and reorganization of lipids, proteins and glycocalyx components at any cell-cell contact.

biophysics↗

bioRxiv2025

Prodan-based solvatochromic probes for polarity imaging of organelles

Solvatochromic probes provide microscopic, structural and functional information on their targeted cellular compartments. In this field, the challenge lies in designing probes that are both sufficiently sensitive to environment, and specific in their subcellular localization. Here, we design Prodan-based polarity probes targeting the following organelles: mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and lipid droplets. The new probes provide robust organelle targeting, except for the mitochondrial probe, whose targeting ability is cell type dependent. Due to operating range of Prodan fluorophore in the UV-Blue region, our probes can be easily combined with other fluorescent tags in the visible range. Therefore, polarity of sub-cellular compartments can be studied together with additional fluorescent reporters. We used these probes to show the polarity of organelle membranes in healthy cells and under starvation condition where we observed organelle-specific polarity remodeling. These probes are important addition to the repertoire of smart cellular probes and will find critical use in understanding spatiotemporal regulation of cellular physiology.

biophysics↗

bioRxiv2025

Image-based screens identify regulators of endogenous Dvl2 biomolecular condensates

Dishevelled (Dvl) proteins are essential transducers in Wnt signaling pathways, which have been implicated in development, stem cell maintenance, and human diseases such as cancer. Several studies have shown that Dvl proteins form dynamic biomolecular condensates. However, how cellular signals and cell states influence the formation of biomolecular condensates remains poorly understood. Here, we analyzed cells with endogenous Dvl2 condensates using image-based cell sorting in combination with phosphoproteomics and identified protein enrichment for Wnt/PCP signaling and the G2/M cell cycle transition. We then performed an image-based high-throughput screen to identify small molecule kinase inhibitors that affect Dvl2 liquid-liquid phase separation. Strikingly, CK1{delta}/{varepsilon} inhibition blocked Dvl2 condensate formation. Its effect on Wnt signaling was modulated in genetic epistasis experiments with loss-of-function alleles of APC, Axin1, and MCC. Our study highlights the interplay between post-translational modifications and condensate dynamics, opening new avenues for research on their role in cellular signaling and disease intervention.

cell biology↗

bioRxiv2024

VISION -- an open-source software for automated multi-dimensional image analysis of cellular biophysics

Environment-sensitive probes are frequently used in spectral/multi-channel microscopy to study alterations in cell homeostasis. However, the few open-source packages available for processing of spectral images are limited in scope. Here, we present VISION, a stand-alone software based on Phyton for spectral analysis with improved applicability. In addition to classical intensity-based analysis, our software can batch-process multidimensional images with an advanced single-cell segmentation capability and apply user-defined mathematical operations on spectra to calculate biophysical and metabolic parameters of single cells. VISION allows for 3D and temporal mapping of properties such as membrane fluidity and mitochondrial potential. We demonstrate the broad applicability of VISION by applying it to study the effect of various drugs on cellular biophysical properties; the correlation between membrane fluidity and mitochondrial potential; protein distribution in cell-cell contacts; and properties of nanodomains in cell-derived vesicles. Together with the code, we provide a graphical user interface for facile adoption. We anticipate that VISION will find a broad range of applications in different fields of biology, spanning from molecular and tissue biology to immunology and biophysics. Summary statementVISION, an open-source software, enables high throughput and correlative analysis of cellular biophysical properties.

biophysics↗

bioRxiv2023

Quantifying fluorescence lifetime responsiveness of environment sensitive probes for membrane fluidity measurements

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and to avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as degree of lipid saturation, cholesterol content, double bond position and configuration and phospholipid headgroup. As comparison, we investigated lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime, but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g. cholesterol). While the lifetime of Flipper is particularly sensitive to membrane cholesterol content, NR12S and NR12A lifetime is moderately sensitive to both cholesterol content and lipid acyl chains. Moreover, all probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission-wavelength dependency results in varying lifetime resolution at different spectral regions, highly relevant for FLIM data acquisition. Our data provides valuable insights on how to perform FLIM with these probes and highlights both their potential and limitations.

biophysics↗

bioRxiv2023

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that act as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings reveal a novel glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

cell biology↗

bioRxiv2022

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties

The plasma membrane serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades several environment-sensitive probes have been popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we studied the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration, phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

biophysics↗