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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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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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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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Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane
Doudová, Lenka ; Malínský, Jan (advisor) ; Vopálenská, Irena (referee)
Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an important role in response to various environmental stresses. Sur7 and Nce102 are transmembrane MCC proteins of unknown function. Sur7 is most likely a structural protein within MCC, Nce102 is probably important in regulation of kinases associated with MCC/eisosomes. Key words: biological membrane, membrane microdomains, eisosome, S. cerevisiae, Sur7, Nce102
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The effect of morphine on the distribution of signaling molecules of the opioid system in lipid rafts prepared from rat heart
Ladislav, Marek ; Novotný, Jiří (advisor) ; Rudajev, Vladimír (referee)
Morphine is an opioid agonist, which can exert cardioprotective effects under certain conditions. Lipid rafts are considered important platforms for membrane organization of signaling proteins and, therefore, these structures could play a role in the effects of morphine, which acts through the opioid receptors. The aim of this thesis was to investigate the distribution of the main components of the opioid receptor and Gi/o-mediated signaling pathway in lipid rafts isolated from rat myocardium, which was affected by various doses of morphine. Because we used different isolation techniques with different solubilization agents (Triton X-100, CHAPS, cholate and sodium carbonate) for preparation of lipid rafts, it was of interest to characterize more closely these preparations. Another aim of this study was to investigate how different methods of isolating these structures affect activity of the key target enzyme of the opioid signaling pathway, i.e. adenylyl cyclase. The presence of signaling molecules of the Gi/o/AC pathway of the opioid system in membrane rafts was confirmed and the distribution of selected proteins was dependent on the type of extractant. We also observed the effect of morphine on the localization of proteins in lipid rafts. Different extractants provided different degree of...
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The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling
Hrdinka, Matouš ; Drbal, Karel (advisor) ; Černý, Jan (referee) ; Kalina, Tomáš (referee)
Dissertation summary The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling Matouš Hrdinka How do the plasma membrane microdomains and transmembrane adaptor proteins (TRAPs) influence the outcome of immunoreceptor signaling? These have been the important questions of molecular immunology. In spite of the years of intensive research, these problems remain incompletely understood. The plasma membrane is a highly dynamic heterogeneous bilayer spontaneously organized into microdomains of various size, composition, and lifetime. The lipid rafts are one example of such microdomains and have been implicated in many biological processes, including immunoreceptor signaling. Because rafts are enriched in many signaling proteins, they are believed to function as platforms for signal initiation and propagation. The TRAPs are important organizers and regulators of immunoreceptor signaling. For example, LAT is indispensable in T cell receptor (TCR) signaling and T cell development, PAG for the regulation of Src family tyrosine kinases (SFKs), and NTAL is a multifunctional negative and positive regulator. The presence of these TRAPs in lipid rafts seems to be crucial for their functions, however, is still a matter of debate. Moreover, other so far...
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