Marek Cebecauer

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

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Martin Hof

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Piotr Jurkiewicz

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Tomáš Chum

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Lukasz Cwiklik

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Tomáš Brdička

Czech Academy of Sciences, Institute of Molecular Genetics

Daniela Glatzová

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Zuzana Kvíčalová

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

RŠ

Radek Šachl

Czech Academy of Sciences

Marie Olšinová

Charles University

Václav Hořejšı́

Czech Academy of Sciences, Institute of Molecular Genetics

Cooperative Institutions

Charles University

Czech Academy of Sciences

Czech Academy of Sciences, J. Heyrovský Institute of Physical Chemistry

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

General University Hospital in Prague

University of Cambridge

Czech Academy of Sciences, Institute of Microbiology

University of Lausanne

Centre National de la Recherche Scientifique

Ludwig Cancer Research

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CD8+ Cytotoxic T Lymphocyte Activation by Soluble Major Histocompatibility Complex-Peptide Dimers

Journal of Biological Chemistry (2005)

Marek Cebecauer Philippe Guillaume Silke Mark Olivier Michielin Nicole Boucheron

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Histocompatibility

Mixed lymphocyte reaction

10.1074/jbc.m500654200

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Citations (59)

Innentitelbild: A Rotational BODIPY Nucleotide: An Environment‐Sensitive Fluorescence‐Lifetime Probe for DNA Interactions and Applications in Live‐Cell Microscopy (Angew. Chem. 1/2016)

Angewandte Chemie (2015)

Dmytro Dziuba Piotr Jurkiewicz Marek Cebecauer Martin Hof Michal Hocek

Die Fluoreszenzlebensdauer von Bodipy-Fluorophoren reagiert empfindlich auf die Viskosität ihrer Umgebung. In der Zuschrift auf S. 182 ff. beschreiben M. Hof, M. Hocek et al, dass ein mit Bodipy verknüpftes Nucleotid, enzymatisch in DNA eingebaut, Sonden ergibt, die auf das Binden von Proteinen mit einer Zunahme der Lebensdauer von 0.5 auf 2.2 ns reagieren. Die DNA-Sonden bilden Lipoplexe, die die Sonden in lebende Zellen transportieren, in denen eine starke Abhängigkeit der Fluoreszenzlebensdauer von der lokalen Mikroumgebung nachgewiesen wurde.

BODIPY

10.1002/ange.201510985

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Citations (8)

Approach to map nanotopography of cell surface receptors

Communications Biology (2022)

Christian Franke Tomáš Chum Zuzana Kvíčalová Daniela Glatzová Gregor Jörg Gentsch

Abstract 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.

Nanotopography

10.1038/s42003-022-03152-y

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Citations (11)

Lipids and proteins in membranes: Fromin silicotoin vivo

Molecular Membrane Biology (2012)

Marek Cebecauer

The cell can be called an elementary unit of the living world. In order to maintain its vital functions, metabolic, signalling and other important processes need to be efficient and robust. One mec...

10.3109/09687688.2012.710344

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Citations (1)

The Motif Orientation Matters: Structural Determinants of TEAD1 Recognition of Genomic DNA

SSRN Electronic Journal (2020)

Růžena Filandrová Karel Vališ Jiří Černý Josef Chmelı́k Lukáš Slavata

Transcription enhancer activator domain (TEAD) transcription factors mediate gene expression regulation through interactions with M-CAT motif. They play an important role during development of nearly all eukaryotic organisms and control expression of multiple genes involved in cell proliferation, differentiation or apoptosis. Moreover, activity of TEAD transcription factors is deregulated in several types of cancers. To study the interaction of TEAD1 with M-CAT motifs originating from regulatory regions of human genes (CTGF, SRF, C-MYC and GLUT1), the dissociation constant of each complex was determined by using fluorescence anisotropy assays. Structural mass spectrometry techniques, molecular docking and single-molecule Förster resonance energy transfer enabled structural characterization of these complexes. Chromatin immunoprecipitation and quantitative PCR analysis validated our in-vitro results. The data also indicate that the affinity of TEAD1-DBD for cognate DNA strongly depends on the orientation of the M-CAT motif. Although, the protein-DNA interaction interface is similar when M-CATs are oriented in either the 5’-CCTTA-3’ or 5’-ATTCC-3’ direction, the former leads to approximately 10× higher affinity. Additionally, the 5’-CCTTA-3’ orientation produced a lower TEAD1 occupancy in Jurkat cells, thus suggesting that the low-affinity binding sites present in the human genome may possess biological relevance which is likely associated with its transcriptional regulation.

Motif (music)

Structural motif

10.2139/ssrn.3640821

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Evaluation of lipid peroxidation by the analysis of volatile aldehydes in the headspace of synthetic membranes using selected ion flow tube mass spectrometry

Rapid Communications in Mass Spectrometry (2018)

Violetta Shestivská Marie Olšinová Kristýna Sovová Jiřı́ Kubišta David Smith

Rationale Oxidative stress of cell membranes leads to a number of pathological processes associated with some diseases and is accompanied by the release of volatile aldehydes, which potentially can be used as biomarkers. Thus, the aim was to investigate peroxidation of defined synthetic membranes by direct quantitative analysis of volatile aldehydes. Methods The concentration spectra of volatile compounds present in the headspace of synthetic membranes under peroxidation stress and following mechanical stress due to sonication were obtained using solid‐phase microextraction (SPME) in combination with gas chromatography/mass spectrometry (SPME‐GC/MS) and selected ion flow tube mass spectrometry (SIFT‐MS). The focus was on the direct, real‐time quantification of volatile aldehydes. In addition, the total aldehydes in the aqueous membrane suspensions were quantified using the thiobarbituric acid reactive substances (TBARS) method. Results Propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde were detected and quantified in the humid headspace of the media containing the synthetic membranes following peroxidation. The composition and concentration of these saturated aldehydes strongly depend on the representation of unsaturated fatty acids in the liposomes. Some protective effect of cholesterol was observed especially for membranes peroxidised by Fenton reagents and after application of a mechanical stress. Conclusions This study demonstrates that peroxidation of model synthetic membranes in vitro can be tracked in real time using direct quantification by SIFT‐MS of several specific aldehydes in the headspace of the membrane suspensions. Cholesterol plays an important role in retaining membrane structure and can indirectly protect membranes from lipid peroxidation.

Thiobarbituric acid

TBARS

10.1002/rcm.8212

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Citations (13)

HeliDye1: helquat fluorogenic probe specific for AT-rich DNA duplexes

Research Square (Research Square) (2022)

Erika Kužmová Vishwas N. Joshi Jaroslav Kozák Lukáš Severa Lucie Bednarova

Abstract DNA-binding fluorescent probes are useful tools in cell biology for various applications enabling visualisation and quantification of DNA. Herein, we report on HeliDye1 , a novel cationic helicene-like DNA probe, and its versatile usability. HeliDye1 exhibits fluorescence exclusively in the presence of DNA duplexes containing AT base pairs and (contrary to any recent DNA probe) it remains non-fluorescent in the presence of single-stranded DNA and RNA. Favourable chemical properties of HeliDye1 allowed its use in super-resolution microscopy and DNA quantification using flow cytometry. We present five more applications of HeliDye1 . Viscosimetry showed that HeliDye1 is a minor groove binder. HeliDye1 interacts with double-stranded DNA as an induced (P) -enantiomer, which we confirmed by ECD spectroscopy and DFT calculations. ECD spectroscopy suggested the formation of HeliDye1 dimers. MD simulations predicted the structures of (P) -HeliDye1 dimers inside DNA and showed their stability in AT-rich sequences and instability in GC-rich sequences.

10.21203/rs.3.rs-737372/v1

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Citations (0)

Motif orientation matters: Structural characterization of TEAD1 recognition of genomic DNA

Structure (2020)

Růžena Filandrová Karel Vališ Jiří Černý Josef Chmelı́k Lukáš Slavata

Sequence motif

Structural motif

Jurkat cells

10.1016/j.str.2020.11.018

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Citations (3)

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

Open Biology (2024)

Federica Scollo Carmelo Tempra Hüseyin Evci Miguel Riopedre-Fernández Agnieszka Olżyńska

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 prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM–membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role—it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the 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.

Phosphatidylethanolamine

Membrane Lipids

Second messenger system

10.1098/rsob.240067

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Citations (1)

Phenotypic Effects of CD3ζ Targeting into Glycosphingolipid-Enriched Membrane Microdomains (GEMs) of T Cells

Biochemical and Biophysical Research Communications (2000)

Marek Cebecauer Jan Černý

Jurkat cells

10.1006/bbrc.2000.2677

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Citations (5)