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On the origin of receptor microclusters on T cells
No full textT cells play an important role in both acquired and innate immunity. T cell receptors recognize antigens presented by MHC glycoproteins on cellular surfaces. The binding of the antigen to the T-cell receptor triggers activation signals. This leads to T-cell receptor clustering to microclusters and immunological synapse generation. The IS plays an important role in signalization, co-stimulation, T-cell activation and receptor degradation. This thesis is focused on the process of the T-cell receptor microclusters and immunological synapse formation and how the development in fluorescence microscopy improved our insight into these processes
Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?
No full text<p>Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?:</p>
<p><br>This data set contains all the experimental raw data, analysis and source files for the final figures reported in the manuscript: "Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?". It is divided into five (1-5) zipped folders, named as the technique used to obtain the data. Each of them, where applicable, consists of three different subfolders (raw data, analysed data, final graph). Read below for more details. </p>
<p>1) ConfocalMicroscopy</p>
<p> 1a) Raw_Data: the raw images are reported as .dat and .tif formats, divided into folders (according to date first yymmdd, and within the same day according to composition). Each folder contains a .txt file reporting the experimental details </p>
<p> 1b) GUVs_Statistics<br> - GUVs_Statistics.txt explains how we generated the bar plot shown in Fig. 1E</p>
<p> 1c) Final_Graph<br> - Figure_1B_1D.png is the figure representing figure 1B and 1D<br> - Figure1E_%ofGUVswithCaMAdsorbptions.csv is the source file x-y of the bar plot shown in figure 1E (% of GUVs which showed adsorption of CaM over the total amount of measured GUVs) <br> - Where_To_Find_Representative_Images.txt states the folders where the raw images chosen for figure 1 can be found </p>
<p>2) FCS<br> <br> 2a) Raw_Data: <br> - 1_points: .ptu files <br> - 2_points: .ht3 files <br> - Raw_Data_Description.docx which compositions and conditions correspond to which point in the two data sets<br> <br> 2b) Final_Graphs:<br> - Figure_2A.xlsx contains the x-y source file for figure 2A</p>
<p> 2c) Analysis: <br> - FCS_Fits.xlsx outcome of the global fitting procedure described in the .docx below (each group of points represents a certain composition and calcium concentration, read the Raw_Data_Description.docx in the FCS > Raw_Data)<br> - Notes_for_FCS_Analysis.docx contains a brief description of the analysis of the autocorrelation curves</p>
<p>3) GPLaurdan<br> <br> 3a) Raw Data: all the spectra are stored in folders named by date (yymmdd_lipidcomposition_Laurdan) and are in both .FS and .txt formats </p>
<p> 3b) GP calculations: contains all the .xlsx files calculating the GP values from the raw emission and excitation spectra</p>
<p> 3c) Final_Graphs<br> - Data_Processing_For_Fig_2D.csv contains the data processing from the GP values calculated from the spectra to the DeltaGP (GP with- GP without CaM) reported in fig. 2D<br> - Figure_2C_2D.xlsx contains the x-y source file for the figure 2C and 2D</p>
<p>4) LiveCellsImaging </p>
<p> 3a) Intensity_Protrusions_vs_Cell_Body: <br> - contains all the .xlsx files calculating the intensity of the various images. File renamed by date (yymmdd) <br> - All data in all excel sheets gathered in another Excel file to create a final graph </p>
<p> 3b) Final_Graphs<br> - Figure_S2B.xlsx contains the x-y source file for the figure S2B</p>
<p>5) LiveCellImaging_Raw_Data: it contains some of the images, which are given in .tif. They are divided by date (yymmdd) and each contains subfolders renamed by sample name, concentration of ionomycin. Within the subfolders, the images are divided into folders distinguishing the data acquired before and after the ionomycin treatment and the incubation time.</p>
<p> </p>
<p>6) 211124_BioCev_Imaging_1 folder has the .jpg files of the time laps, these are shown in fig 1A and S2.</p>
<p>7) 211124_BioCev_Imaging_2 and 8) 211124_BioCev_Imaging_3 contain the images of HeLa cells expressing EGFP-CaM after treatment with ionomycin 200 nM (A1) and 1 uM (A2), respectively. </p>
<p><br>9) SPR</p>
<p> 9a) Raw Data: <br> - SPR_Raw_Data.xlsx x/y exported sensorgrams <br> - the .jpg files of the software are also reported and named by lipid composition</p>
<p> 9b) Final_Graph: <br> - Fig.2B.xlsx contains the x-y source file for the figure 2B</p>
<p> 9c) Analysis<br> - SPR_Analysis.xlsx: excel file containing step-by-step (sheet by sheet) how we processed the raw data to obtain the final figure (details explained in the .docx below)<br> - Analysis of SPR data_notes.docx: read me for detailed explanation</p>
The role of structural motifs in the localisation of T-cell plasma membrane proteins
No full textPlasma membrane of T cells is abundant in diverse receptors and other molecules orchestrating immune responses. Numerous studies demonstrate that the localisation of proteins in the cell is non-random and that mislocalisation either in the context of plasma membrane at nanoscale or with respect to the cell interior can lead to the protein malfunction and subsequent aberrant T- cell response. In my first Ph.D. project we focused mainly on the role of the transmembrane domain length and amino acid composition, proximal sequences and the presence or absence of palmitoylation on the localisation of transmembrane adaptor proteins LAT, PAG and NTAL in T cells. We showed that plasma membrane localisation of PAG and NTAL is controlled by the amino acid composition of their TMD and is palmitoylation independent. We propose that NTAL localisation to the plasma membrane is, despite its suboptimal length, facilitated by the electrochemical asymmetry of its TMD. Among transmembrane adaptor proteins, LAT was the most interesting one. Dependency of LAT plasma membrane localisation on palmitoylation in combination with unusual amino acid composition of its TMD led us to investigate it in a separate project. My first author Ph.D. project was thus to elucidate the role of highly conserved helix-breaking amino acids,..
Characterization of closed mitosis in the fission yeast Schizosaccharomyces pombe with perturbed lipid metabolism
No full text[EN] The division of an eukaryotic cell is mediated by the process of mitosis. It is a complex cellular process which needs to be highly regulated. In contrast to the mammalian open type of mitosis when nuclear envelope is disassembled, fission yeast Schizosaccharomyces pombe undergoes closed mitosis inside the intact nuclear compartment. Cell nucleus undergoes morphological changes as a common sphere-shaped nucleus stretches upon mitotic spindle activity forming typical dumbbell structure. Further tension results in the separation of two daughter nuclei. Such extensive changes in the nuclear envelope surface demand a sufficient supply of membrane phospholipids. Cells with perturbed lipid metabolism are unable to meet such a demand and the mitotic division in these cells usually results as a catastrophic mitotic event or CUT (Cell Untimely Torn) phenotype. Moreover, recent studies show genetic interactions between the deletions of the lipid gene regulator cbf11 and factors maintaining the centromere chromatin structure. Surprisingly, rescue of CUT phenotype has been recently reported after the deletion of several factors contributing to the centromeric H3K9 epigenetic modifications in the cells lacking the transcription factor Cbf11. Here we show no rescue of CUT phenotype after the deletion of..
Bioorthogonal labelling of surface receptors on living lymphocytes
No full textThe surface of cells displays high heterogeneity on chemical and geometrical levels. To understand the function of cells, we need to pay attention to the morphological features formed at the plasma membrane. To study cell surface with molecular specificity, there are plenty of imaging methods starting with the conventional wide-field microscopy through confocal microscopy, ending with super-resolution fluorescence microscopies and electron microscopies. Super-resolution microscopy studies conducted on the fixed cells provide detailed steady-state data about the cell surface nanoscopic organisation and distribution of molecules at the morphological structures. However, since cells are parts of living organisms and constantly change their properties in time and space, the information about dynamics of cellular structures and motility of molecules remains hidden when using this approach. Live-cell compatible methods are required to study dynamic changes of molecules at the single-molecule level. In this study we are focusing on the distribution and dynamics of molecules CD2 and CD4 expressed on the surface of non-stimulated T cells. The main aim of this thesis was to develop a novel method for live-cell imaging and single-molecule tracking of membrane-bound proteins in 3D and at nanoscale. With such a..
Impact of membrane properties on clustering of transmembrane peptides
No full textUnfolded protein response (UPR) is a complex cellular mechanism induced upon ER stress caused by various environmental factors. Single spanning signal transducers of UPR were reported to recognise also lipid-induced ER stress. Studies of these transducers, namely PERK and IRE1 uncovered that they can sense change in membrane properties and activate themselves by clustering. Moreover, signal transducer IRE1 retained ability to sense changes in the membrane properties with TMD exchanged for a polyLeu α-helix. It was thus unclear what mechanism drives lipid-induced UPR via IRE1. We employed model membrane system in form of LUVs, where properties of membranes can be readily altered by specific lipid composition. As a simplified model of the UPR signal transducers in the ER, synthetic transmembrane peptides with polyLeu core were used. Dynamic light scattering (DLS) has been used for qualitative and semi-quantitative analysis of LUVs. Clustering of synthetic peptides was determined by time resolved anisotropy of fluorescence. DLS results demonstrate successful formation of vesicles with a desired size in all planned composition. On the contrary to the studies in living cells, the presence of cholesterol or palmitic acid in model membranes did not induce the aggregation of transmembrane peptides...
Regulation of LAT trafficking to the plasma membrane
No full textLinker for activation of T cells is a palmitoylated transmembrane adaptor protein, which is expressed in most of immune cells, but not in immature and mature B cells. It plays an important role in T-cell activation and maturation. LAT is synthesized in the endoplasmic reticulum. Its sorting to the plasma membrane is controlled with various determinants, such as properties of the transmembrane domain and structural motifs in the intracellular part of the protein. Some of those determinants are important for posttranslational modifications, export from the Golgi apparatus and, probably, membrane microdomain targeting, while others interact with COPII machinery and mediate protein export from the endoplasmic reticulum or targeting to endosomes. Keywords: LAT, functional motifs, protein sorting, plasma membrane, Golgi apparatu
Bioortogonální značení povrchových receptorů na nefixovaných lymfocytech
Get PDFThe surface of cells displays high heterogeneity on chemical and geometrical levels. To understand the function of cells, we need to pay attention to the morphological features formed at the plasma membrane. To study cell surface with molecular specificity, there are plenty of imaging methods starting with the conventional wide-field microscopy through confocal microscopy, ending with super-resolution fluorescence microscopies and electron microscopies. Super-resolution microscopy studies conducted on the fixed cells provide detailed steady-state data about the cell surface nanoscopic organisation and distribution of molecules at the morphological structures. However, since cells are parts of living organisms and constantly change their properties in time and space, the information about dynamics of cellular structures and motility of molecules remains hidden when using this approach. Live-cell compatible methods are required to study dynamic changes of molecules at the single-molecule level. In this study we are focusing on the distribution and dynamics of molecules CD2 and CD4 expressed on the surface of non-stimulated T cells. The main aim of this thesis was to develop a novel method for live-cell imaging and single-molecule tracking of membrane-bound proteins in 3D and at nanoscale. With such a...Buněčný povrch vykazuje vysokou heterogenitu na chemické I geometrické úrovni. Abychom porozuměli funkci buněk, musíme věnovat pozornost morfologickým znakům vytvořeným na plazmatické membráně. Pro studium buněčného povrchu s molekulární specifitou existuje spousta zobrazovacích metod, počínaje konvenční wide-field mikroskopií, přes konfokální mikroskopii, konče super-rezoluční fluorescenční mikroskopií a elektronovou mikroskopií. Studie využívající super-rezoluční mikroskopii prováděné na fixovaných buňkách poskytují podrobná steady-state data o nanoskopické organizaci buněčného povrchu a distribuci molekul v rámci morfologických struktur. Protože jsou však buňky součástí živých organismů a neustále mění své vlastnosti v čase a prostoru, informace o dynamice buněčných struktur a pohyblivosti molekul zůstávají při použití tohoto přístupu skryty. Ke studiu dynamických změn na úrovni jedné molekuly jsou nutné metody kompatibilní s životaschopností buněk. V této studii se zaměřujeme na distribuci a dynamiku molekul CD2 a CD4 exprimovaných na povrchu nestimulovaných T buněk. Hlavním cílem této práce bylo vyvinout novou metodu pro zobrazování živých buněk a sledování jednotlivých molekul membránově-vázaných protein ve 3D a s nanometrovou přesností. Pomocí takového nástroje lze zkoumat dynamiku...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult
Localisation of CD4 coreceptor and its variants in human T cells
No full textCD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the..
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