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Bioorthogonal labelling of surface receptors on living lymphocytes
Paldusová, Kateřina ; Cebecauer, Marek (advisor) ; Benda, Aleš (referee)
The 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...
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Characterization of closed mitosis in the fission yeast Schizosaccharomyces pombe with perturbed lipid metabolism
Hohoš, Patrik ; Převorovský, Martin (advisor) ; Cebecauer, Marek (referee)
[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...
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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The role of structural motifs in the localisation of T-cell plasma membrane proteins
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Brábek, Jan (referee) ; Rozbeský, Daniel (referee)
Plasma 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,...
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Binding proteins of MTMR9
Holšteinová, Aneta ; Doubravská, Lenka (advisor) ; Cebecauer, Marek (referee)
Myotubularins are lipid phosphatases that dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate the position three of the inositol ring. This allows them to regulate the structure of the lipid layer of the membrane compartment. The first member of the family was described in association with a severe hereditary myopathy. From that point on, another thirteen members have been added to the family. The catalytically inactive MTMR9 carrying the conserved mutation in the phosphatase domain regulates the localization of the marker of the early secretory pathway, RAB1A, the cis-Golgi structure and the secretion. MTMR9 interacts with the catalytically active MTMR6 and MTMR8 that specifically localizes and increases their phophatase activity. The aim of this diploma thesis was to find out whether the phenotype observed in cells with altered MTMR9 levels is dependent on the catalytically active phosphatases MTMR6 and MTMR8. We proved the influence of MTMR6 and MTMR8 on the distribution of tranfected RAB1A between the intermediate compartment and the Golgi apparatus. MTMR6 and MTMR8 also take part in regulating the cis-Golgi structure. By the use of two different approaches we did not manage to clarify the influence of MTMR6 and MTMR8 on secretion. Changes in the catalytic...
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Compartmentalisation of surface receptor signalling in T cells
Paldusová, Kateřina ; Cebecauer, Marek (advisor) ; Janušová, Šárka (referee)
The main goal of this thesis is to introduce the topic of compartmentalisation of signalling on the surface of T lymphocytes before and during the T-lymphocyte activation by the contact with an antigen-presenting cell (APC) or a target cell. To understand the compartmentalisation of signalling, the morphology of cell surface needs to be understood first. It is believed that the surface of T cell is magnified with an abundance of membrane protrusions called microvilli. Although, little is known about their inner structure. They may have some structural features different from microvilli on the surface of APC or microvilli of intestinal brush border. These structures are further described and compared with each other along-side with some other non-microvillar membrane protrusions and extensions. The insight into three-dimensional compartmentalisation of signalling molecules in T cells is still in its early days. Some results even contradict each other. This field will require the application of advanced imaging methods in a rather comprehensive way to uncover fine organisation of these molecules before and after encountering stimulatory surface. Key words T-cell signalling, membrane receptors, cell surface morphology, microvilli, microscopy
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On the origin of receptor microclusters on T cells
Ptáček, Antonín ; Cebecauer, Marek (advisor) ; Černý, Jan (referee)
T 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.
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Quantitative fluorescence microscopy techniques to study three-dimensional organisation of T-cell signalling molecules.
Chum, Tomáš ; Cebecauer, Marek (advisor) ; Lánský, Zdeněk (referee) ; Brameshuber, Mario (referee)
10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated with...
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Regulation of LAT trafficking to the plasma membrane
Rakhimbekova, Anastasia ; Cebecauer, Marek (advisor) ; Černý, Jan (referee)
Linker 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 apparatus
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Impact of membrane properties on clustering of transmembrane peptides
Sabó, Ján ; Cebecauer, Marek (advisor) ; Vít, Ondřej (referee)
Unfolded 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....
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