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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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Mechanisms of transmembrane auxin transport in a broader evolutionary context.
Rubešová, Magdaléna ; Petrášek, Jan (advisor) ; Tylová, Edita (referee)
Auxin, represented by the molecule indole-3-acetic acid (IAA), is one of the main phytohormones involved in the regulation of plant development. Its intercellular transport establishes concentration gradients in individual cells that control gene expression and a number of downstream processes. In plants, a complex mechanism for efficient IAA transport has evolved, involving both long-distance transport and intercellular transport within individual tissues. Because our understanding of the auxin transport mechanisms is still incomplete, this thesis attempts to summarize the literature data on all modes of auxin transport across cell membranes that have been recognized to date and places them in a broader evolutionary context. The presence of IAA in many prokaryotic and eukaryotic organisms, together with the similarly wide occurrence of carriers from "auxin efflux carrier" transporter family, evolutionarily related PIN-FORMED-like carriers, points to the possibility that IAA transport may also be evolutionarily very ancient and may functionally derive from more general mechanisms of ions or amino acids.
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Cellular determinants of the distribution of PIN auxin transporters in the plasma membrane
Stelate, Ayoub ; Petrášek, Jan (advisor) ; Mravec, Jozef (referee) ; Nodzynski, Tomasz (referee)
Asymmetric localization of auxin carriers has always attracted the attention of many scientists around the world. However, to address this topic while focusing on the plasma membrane (PM), requires advanced microscopy techniques and knowledge of biophysics and biology. This doctoral work bridges the two disciplines to contribute to our understanding of the dynamics and distribution of PIN-FORMED auxin carriers from tobacco (NtPINs) within the PM. I have developed a novel correlative light electron microscopy (CLEM) method using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). To my knowledge, this is the first effort to achieve a correlation between immunofluorescence and electron microscopy imaging of plant integral PM proteins. As I have shown, individual NtPINs are organized differently within the PM. Dynamic analyses that quantify individual nanodomains' diffusion rates allowed me to show that NtPINs have constraints behavior with different diffusion rates. I have investigated the role of the cell wall and cytoskeleton in the organization and dynamics of NtPINs. Using pharmacological treatments, I have shown that they differentially affect the mobility and organization of NtPINs within the PM. Complete removal of the...
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The role of actin cytoskeleton in the targeting of auxin carriers to the plasma membrane.
Kebrlová, Štěpánka ; Petrášek, Jan (advisor) ; Pernisová, Markéta (referee)
Auxin plays an important morphogenic role in plant development, mainly through its effect on gene expression, but also through a number of faster processes that are directly dependent on its concentration. Therefore, in many plant tissues, directional auxin transport using specific transporters in the plasma membrane, is important for the coordination of morphogenic stimuli. The amount of auxin carriers in the plasma membrane directly affects the resulting auxin concentration inside the cell. Although the localization of auxin transporters and their abundance in the plasma membrane could be determined primarily by the actin cytoskeleton and its involvement in vesicle transport processes, this relationship is currently still unclear. Therefore, in this study, we were interested in how the localization and function of auxin transporters is affected when the function of the actin cytoskeleton is affected in a given cell type. To this end, the localization of the auxin transporters PIN3, PIN4, PIN7, and AUX1 was studied in epidermal cells of cotyledons in young seedlings of Arabidopsis thaliana whose morphogenesis was affected by mutations in subunits of the actin nucleation complex ARP2/3. Crosses of mutants in the ARP2/3 complex subunits with marker lines carrying fluorescently labeled auxin carriers...
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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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The effect of ethinylestradiol on Na+, K+ - ATPase
Kettnerová, Karolína ; Svoboda, Petr (advisor) ; Driák, Daniel (referee)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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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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Caveolae and caveosoms
Galica, Tomáš ; Černý, Jan (advisor) ; Forstová, Jitka (referee)
Caevolae are remarkably stable structures at the plasma membrane. They form specific domains distinct in lipid composition from the rest of plasma membrane. Many diverse functions are assigned to Caevolae. They play role in modulation of cellular surface, signalization and well regulated endocytosis. Caveosomes suppose to be large intracellular vesicular structures potentialy new membrane organels. They are derived from internalized caveolae. Tohether with caveolae they are proposed to form a separeted system of intracellular vesicles. However recent evidence suggests that caveolae can fuse with endosomes immediately after internalization. If this is true, then the system of vesicles derived from caveolae, including caveosomes, can be considered a regular component of endosomal system. Isolation of caveosomes from endosomes has been seen mainly in experiments where polyomavirus SV40 was used. Thus the question, if this isolation is not just a result of SV40 infection, arises. It has been shown recently that SV40 virus is capable of inducing caveosome-like structures even in the absence of caveolae. Consequently existence and properties of caveosomes are being questioned. The problem of high importance is the genesis of caveosomes and their existence in SV40 non-infected cells. In this thesis...
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AUXIN BINDING PROTEIN 1 (ABP1) and its role in the auxin management in plant cells
Čovanová, Milada ; Zažímalová, Eva (advisor) ; Lüthen, Hartwig (referee) ; Reinöhl, Vilém (referee)
Conclusions The role of AUXIN BINDING PROTEIN 1 (ABP1) in the auxin management in plant cells was followed using simplified model material of suspension-cultured cells of tobacco BY-2 line. ABP1 is a putative auxin receptor considered to mediate fast non-genomic responses to auxin and it can be involved in every aspect of the regulation of auxin responses, metabolism and transport. There are four major conclusions that could be made based on the results presented in this thesis: 1) Auxin binding protein 1 mediates both cell division and expansion in tobacco BY-2 cells. In standard cultivation conditions or at lower concentrations of 2,4-D in culture medium, ABP1 overexpression had no detectable impact on cell division, cell elongation or cell growth.. 5- times increased 2,4-D concentration stimulated weakly cell elongation. . Antisense suppression of ABP1 expression resulted in disturbance in both cell expansion and cell division intensity, suggesting that ABP1 is essential for the control of balance between cell division and cell elongation during the growth cycle. ABP1 is localized in endoplasmic reticulum of cells cultivated in standard medium supplemented with 1 μM 2,4-D and it appeared also at the plasma membrane following the IAA application. 2) ABP1 mediates intercellular auxin transport. Cells...
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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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