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Vesicular roles of Arp2/3 nucleation-promoting factors
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Malínský, Jan (referee) ; Befekadu, Asfaw (referee)
F-actin is involved in key aspects of vesicular traffic, such as membrane deformation, tubulation and vesicle motion. Branching of F-actin is mediated by Arp2/3 but this complex must first be activated by so-called nucleation-promoting factors (NPFs). These factors play an essential role in the decision where and when branched actin should form on the membrane surface. The thesis focuses on the mechanisms which underlie localization and activation of NPFs, especially in terms of the phosphoinositide composition of the vesicle membranes. I show that one of the NPFs, the WASH complex, does not exclusively depend on the retromer complex for its membrane anchoring, as previously theorized. Rather, its understudied subunit SWIP enables the complex to independently bind to the membrane. I also present data showing that the WASH complex has essential roles in maintaining lysosomal function. Additionally, I elucidate the function of another NPF known as WHAMM in the ERGIC compartment, showing that it depends on the presence of myotubularin 9 for its ability to form membrane tubules. The thesis improves our understanding of the interface between the actin cytoskeleton and intracellular membrane system.
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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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New interconnections between lipid metabolism and chromatin regulation
Princová, Jarmila ; Převorovský, Martin (advisor) ; Bágeľová Poláková, Silvia (referee) ; Malínský, Jan (referee)
Lipid metabolism has been implicated in changes of chromatin modifications resulting in altered gene expression. Such regulation is important for cellular differentiation or cancer progression, however, the mechanism of how altered metabolic flux leads to targeted changes in chromatin modifications which then regulate gene transcription or heterochromatin maintenance is still poorly understood. We describe that fission yeast cells defective in fatty acid synthesis show increased expression of a subset of stress-response genes. This altered gene expression depends on the SAGA and NuA4 histone acetyltransferases and is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters. Moreover, diminished fatty acid synthesis results in increased cellular resistance to oxidative stress. Additionally, the lipid metabolism mutants display chromatin alterations in centromeres and subtelomeres, regions of constitutive heterochromatin. We propose that changes in lipid metabolism can regulate histone acetylation and transcription of specific stress-response genes, but also lead to more global changes in heterochromatin. And while we clearly see the consequences of increased stress-response genes which result in promoting redox homeostasis, the implications of altered...
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Trk1 Potassium Importers - key transport systems for yeast cell fitness and stress tolerance
Masaryk, Jakub ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Malínský, Jan (referee)
One of the key prerequisites for yeast cell growth is the uptake of essential compounds, such as potassium. Potassium is a vital monovalent cation and its sufficient intracellular concentration is crucial for various processes, for instance: regulation of membrane potential and cell turgor, enzymatic activity, and protein synthesis. A sufficient internal concentration of potassium is also one of the pivotal signals for cell division. However, as also excess of potassium might lead to unfavourable physiological consequences in yeast, such as deacidification of vacuoles and depolarization of plasma membrane, it is imperative for the yeast cells that the whole process of potassium acquisition is a tightly regulated affair, in order to maintain proper potassium homeostasis. In yeast Saccharomyces cerevisiae, uniporter Trk1 is considered a key player in potassium uptake. The presented thesis aimed to provide novel knowledge regarding Trk1, more specifically to study its ability to modify its capacity for potassium uptake, putative regulation by phosphorylation, and involvement in the survival of glucose-induced cell death (GICD). Additionally, potassium-uptake systems in selected non-conventional species were characterized as well. The most distinctive feature of Trk1 is its alleged ability to switch...
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Lipid Membranes at the Nanoscale: Single-Molecule Fluorescence Approach
Koukalová, Alena ; Černý, Jan (advisor) ; Malínský, Jan (referee) ; Benda, Aleš (referee)
The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...
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Quality control in snRNP biogenesis
Roithová, Adriana ; Staněk, David (advisor) ; Malínský, Jan (referee) ; Vomastek, Tomáš (referee)
(English) snRNPs are key components of the spliceosome. During their life, they are found in the cytoplasm and also in the nucleus, where carry out their function. There are five major snRNPs named according to RNA they contain U1, U2, U4, U5 and U6. Each snRNP consists from RNA, ring of seven Sm or LSm proteins and additional proteins specific for each snRNP. Their biogenesis starts in the nucleus, where they are transcribed. Then they are transported into the cytoplasm. During their cytoplasmic phase, the SMN complex forms the Sm ring around the specific sequence on snRNA and cap is trimethylated. These two modifications are the signals for reimport of snRNA into the nucleus, where they accumulate in the nuclear structures called Cajal bodies (CBs), where the final maturation steps occur. There are several quality control points during snRNP biogenesis that ensure that only fully assembled particles reach the spliceosome. The first checkpoint is in the nucleus immediately after the transcription, when the export complex is formed. The second checkpoint is in the cytoplasm and proofreads Sm ring assembly. If the Sm ring formation fails, the defective snRNPs are degraded in the cytoplasm by Xrn1 exonuclease. However, it is still unclear, how the cell distinguishes between normal and defective...
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Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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Analysis of functional interactions of phospholipids in the cell nucleus.
Biddle, Veronika ; Hozák, Pavel (advisor) ; Kaňka, Jiří (referee) ; Malínský, Jan (referee)
(English) Phosphoinositides (PIs) are glycerophospholipids with a negative charge. As components of cell membranes, PIs are involved in membrane and cytoskeletal dynamics, cell movement and signalling, and the modulation of ion channels and transporters. Apart from the cytoplasm, phosphoinositides also localise to the cell nucleus. PIs play a role in crucial nuclear processes, such as DNA transcription, pre-rRNA and pre-mRNA processing, cell differentiation, DNA damage response, or apoptosis. Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are the most abundant phosphoinositides in the cell. However, their exact localisation and function in the nucleus are largely unknown. Here, we describe their localisation at super-resolution level and their involvement in some nuclear processes. PI(4)P is present in nuclear lamina, nuclear speckles and nucleoli, and it forms small foci in nucleoplasm. The majority of nuclear PI(4)P localises to the nucleoplasm, whereas almost 16 % is present in nuclear speckles. On the other hand, the majority of nuclear PI(4,5)P2 localises to nuclear speckles, almost 30 % localises to nucleoplasm and the lesser portion to nucleoli. In the nucleoplasm, PI(4,5)P2 forms small foci called nuclear lipid islets (NLIs). Their core is...
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