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Membrane microdomains in regulation of lipid metabolism
Veselá, Petra ; Malínský, Jan (advisor) ; Hašek, Jiří (referee) ; Zimmermannová, Olga (referee)
The fluid mosaic model described by Singer and Nicolson in 1972 was timeless and remains relevant to understanding the structure, function and dynamics of biological membranes more than fifty years later. From the outset, its authors acknowledged the existence of lateral membrane regions that differ in composition and biological function from their immediate surroundings. Therefore, even contemporary studies demonstrating the existence of many different membrane microdomains do not pose a fundamental challenge to the validity of this model. In particular, research over the last twenty years has shown that a number of cellular processes (nutrient transport, signaling, regulation of nucleic acid metabolism, lipophagy and many others) are linked to membrane microdomains. However, the molecular details of these links remain hidden in many cases. The aim of this work is to find specific links between membrane microdomains and the metabolism of selected lipids. Using the yeast model S. cerevisiae, we document the connection of a specialized plasma membrane microdomain, the membrane compartment of arginine permease Can1 (MCC), to the metabolism of sphingolipids and the mitochondrial anionic phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL). The initial two chapters deal with the elucidation of...
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Dynamics of yeast plasma membrane domains
Oláhová, Dominika ; Zahumenský, Jakub (advisor) ; Vopálenská, Irena (referee)
Plasmatic cellular mebrane consists of specialized domains which differ in shape, function and size. This compartmentalization allows cells to coordinate and separate a large number of diverse processes. Disorders of cell organization at the level of membrane domains are increasingly associated with the expansion of pathological disorders. Microdomains also play a role in the virulence of pathogenic organisms, e.g. some types of yeast. One of the best described microdomains of the yeast plasma membrane is the MCC (membrane compartment of arginine permease Can1), which is stabilized by a protein complex called the eisosome. The composition of the MCC/eisosome is described in detail, the dynamics of the individual components in the basal state (i.e. without stress) has not yet been described. The aim of this thesis is to summarize current knowledge about the dynamics of the proteins of this microdomain and its relationship with other domains of cell membranes through a detailed review of the available literature. And at the same time by using the advanced fluorescence method FRAP (fluorescence recovery after photobleaching) to show in my own experiment that in the basal state there is an exchange of individual Nce102 molecules in the MCC/eisosome microdomain. Key words: yeast, plasma membrane,...
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
Toxin translocation across the cytoplasmic membrane of the eukaryotic cell is a potent virulence factor of bacteria causing disease to eukaryotic organisms. Toxins translocate their domains responsible for the toxic activity inside the cell or create pores in cell membrane allowing the transmembrane traffic of ions, DNA, RNA or proteins. Knowledge of the toxin translocation process enables to characterize the mechanism and also the properties of the pore-forming toxin. Some of these toxins have been described in such a detail that were changed using site-directed mutagenesis and can serve as tools for characterization of the translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane through the pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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Characterization of Arabidopsis thaliana FLOTILLINs and HYPERSENSITIVE INDUCED RESPONSE proteins - dynamics, interactions and functions
Daněk, Michal ; Martinec, Jan (advisor) ; Baluška, František (referee) ; Ovečka, Miroslav (referee)
This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...
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Effect of cholesterol depletion on signalling cascade initiated with receptors coupled to G protein class Gq/G11
Ostašov, Pavel ; Svoboda, Petr (advisor) ; Teisinger, Jan (referee) ; Hof, Martin (referee)
Membrane domains are an important structure in plasamatic membrane. They concentrate various signaling molecules. Their main structural component is cholesterol and by its removal the membrane domains are disrupted. The aim of our work was to examine the effect of cholesterol depeletion on signaling initiated thyreothropin releasing hormone (TRH). Although its signaling cascade is located within membrane domains the receptor itself is not. We showed that cholesterol depletion by -cyclodextrin caused release of Gq/11 proteins and caveolin 2 from membrane domains. We also discovered that cholesterol depletion decreases potency of TRH to activate G proteins as well as induction of release of intracellular Ca2+ In the last part we investigated the effect of disruption of the cell membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility and internalization in HEK293 cells stably expressing TRH-R-eGFP fusion protein. CLSM studies indicated that the internalization of receptor molecules initiated by TRH stimulation was significantly attenuated. The detailed analysis of recovery of TRH-R-eGFP fluorescence in bleached spots of different sizes indicated that cholesterol depletion results in an increase of overall receptor mobility. We suggest that migration of...
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Changes in domain organization of the plasma membrane in the stress response
Vaškovičová, Katarína ; Malínský, Jan (advisor) ; Zimmermannová, Olga (referee) ; Cvačková, Zuzana (referee)
MCC/eisosomes are yeast plasma membrane microdomains that respond to changes in both extracellular and intracellular conditions and activate important stress-related signaling pathways. In this study, we investigated the function of MCC/eisosomes under the conditions of chronic glucose depletion. We found that MCC/eisosomes regulate mRNA decay under these conditions. Specifically, we demonstrated that the sequestration of the evolutionarily conserved Xrn1 exoribonuclease at MCC/eisosomes leads to the attenuation of its enzymatic activity. Modulation of activity by the enzyme localization may represent a novel and effective mechanism in regulation of biochemical pathways. Moreover, our results suggested that an MCC protein Nce102 might play a role in vacuolar fusion and lipid droplets degradation. We demonstrated that prolonged chronic glucose depletion induces the translocation of Nce102 from MCC to sterol-enriched microdomains in the vacuolar membrane. Deletion mutants lacking Nce102 and its functional homologue Fhn1 exhibited significant delay in vacuole maturation and in turnover of a lipid droplet marker Erg6. The function of MCC/eisosomes in the stress response have been demonstrated in many fungal species. Similar to the microdomain function, also individual protein components of...
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Characterization of Arabidopsis thaliana FLOTILLINs and HYPERSENSITIVE INDUCED RESPONSE proteins - dynamics, interactions and functions
Daněk, Michal ; Martinec, Jan (advisor) ; Baluška, František (referee) ; Ovečka, Miroslav (referee)
This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...
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Mechanism of cytokinin transport across plasma membrane
Nedvěd, Daniel ; Ryšlavá, Helena (advisor) ; Dračínská, Helena (referee)
Cytokinins are one of major groups of phytohormones, signaling molecules participating on the crucial physiological processes of plants. Regulation of their function is mostly associated with metabolic changes, recent identification of membrane proteins AtABCG14 and AtPUP14 as selective cytokinin transporters has brought up the possibility of the regulation of cytokinin signalization via polar membrane transport, though. Using the accumulation of radioactively labeled cytokinins in transgenic BY-2 cell culture, we studied the kinetics of cytokinin transport via AtABCG14 and AtPUP14 transporters at cellular level and possible changes of cytokinin metabolism linked to elevated expression of these transporters. Elevated expression of AtPUP14 transporter led to elevated accumulation of labeled cytokinins in form of both ribosides and free bases, while it had no effect on metabolism of labeled cytokinins. Elevated expression of AtABCG14 had no effect on accumulation of labeled cytokinins in BY-2 cells. (In Czech) 1
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Endocytosis in plants
Svitáková, Lenka ; Krtková, Jana (advisor) ; Hála, Michal (referee)
Endocytosis is a process typical for eukaryotic cells. It is a mechanism that ensures invagination of the plasma membrane which leads to the creation of a membrane vesicle with specifically selected cargo. The vesicle is then internalized by the cell. Endocytosis has a crucial role in plant growth and development not only thanks to the reception of nutrients from the extracellular space but also by having a huge impact on the membrane composition which influences cellular signalling. There are few types of endocytosis known in plants. They differ from each other by the molecular machinery which ensures their process. The most studied type in plants is the clathrin-mediated endocytosis. Clathrin-coated vesicle formation proceeds through five stages. However, there are less details known about it in plant cells than in animal cells. In the first part of my bachelor thesis I describe recent knowledge of this topic and the most important participants in this process. I also mention the mechanisms of regulations that plant cells use to coordinate clathrin-mediated endocytosis. In the second part I summarize recently discovered types of endocytosis in plants that are independent from clathrin. These are endocytosis associated with microdomains and fluid- phase endocytosis. The details of these endocytic...
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The role of PIP5K family kinases in plasma membrane remodeling
Apolínová, Kateřina ; Macůrková, Marie (advisor) ; Lišková, Petra (referee)
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is the enzyme responsible for the production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which has long been known as a precursor of two important second messengers, diacylglycerol and inositol trisphosphate. However, PI(4,5)P2 also acts as a second messenger in its own right and regulates many processes occurring on the plasma membrane such as endo- and exocytosis, actin cytoskeleton remodeling, and the formation of cell-cell contacts. The action of PIP5K is carefully spatially and temporally regulated in order to form localized pools of PI(4,5)P2 crucial for its many roles in a wide variety of cell processes. This bachelor's thesis focuses on the description of regulatory mechanisms that control PIP5K activity in vivo and on its physiological functions at the plasma membrane.
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