Biology subjects

Cebecauer, M.

Publications and source records attributed to Cebecauer, M..

6 recordsLinked to original sources

bioRxiv2024

Unraveling the GM1 specificity of Galectin-1 binding to lipid membranes

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1s activity has been suggested to be connected to two molecular concepts, which are however lacking experimental proof: a) enhanced binding affinity of Gal-1 towards membranes containing monosialotetrahexosylganglioside (GM1) over disialoganglioside GD1a and b) cross-linking of GM1s by homodimers of Gal-1. We provide evidence about the specificity and the nature of Gal-1 interaction with model membranes containing GM1 or GD1a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 binds indeed specifically to GM1, and not to GD1a, when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 10 M). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM1 cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM1 and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM1/Gal-1 equilibria towards the membrane-bound homodimeric Gal-1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/614102v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@c4ff01org.highwire.dtl.DTLVardef@141c82eorg.highwire.dtl.DTLVardef@1bd7c0borg.highwire.dtl.DTLVardef@11af49e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Cell surface morphology mimicking nano-bio platform for immune cell stimulation

Studying the complex realm of cellular communication and interactions by fluorescence microscopy requires sample fixation on a transparent substrate. To activate T cells, which are pivotal in controlling the immune system, it is important to present the activating antigen in a spatial arrangement similar to the nature of the antigen-presenting cell, including the presence of ligands on microvilli. Similar arrangement is predicted for some other immune cells. In this work, immune cell-stimulating platform based on nanoparticle-ligand conjugates have been developed using a scalable, affordable, and broadly applicable technology, which can be readily deployed without the need for state-of-the-art nanofabrication instruments. The validation of surface biofunctionalization was performed by combination of fluorescence and atomic force microscopy techniques. We demonstrate that the targeted system serves as a biomimetic scaffold on which immune cells make primary contact with the microvilli-mimicking substrate and exhibit stimulus-specific activation.

cell biology↗

bioRxiv2023

Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?

Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and Molecular Dynamics simulations proves unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. Lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but its binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role - it forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer to increase exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

biophysics↗

bioRxiv2020

The role of prolines and glycine in transmembrane domain of LAT

LAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

cell biology↗

bioRxiv2019

Unraveling nanotopography of cell surface receptors

Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

cell biology↗

bioRxiv2016

The impact of a simple α helical transmembrane peptide on lipid membrane dynamics

Transmembrane domains integrate proteins into cellular membranes and support their function. The capacity of these prevalently a-helical structures in mammals to influence membrane properties is poorly understood. Combining experiments with molecular dynamics simulations, we provide evidence that helical transmembrane peptides with their rough surface reduce lateral mobility of membrane constituents. The molecular mechanism involves trapping of lipid acyl chains on the rough surface and segregation of cholesterol from the vicinity of peptides. The observations are supported by our toy model indicating strong effect of rough objects on membrane dynamics. Herein described effect has implications for the organization and function of biological membranes, especially the plasma membrane with high cholesterol content.

biophysics↗