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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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The role of Nuclear Phosphatidylinositol 4,5 bisphosphate in RNA Polymerase II Transcription
Balaban, Can ; Hozák, Pavel (advisor) ; Šebesta, Marek (referee) ; Castano, Enrique (referee)
(in English) The phosphatidylinositols are a subclass of glycerophospholipids with their inositol head group linked to the diacylglycerol backbone. The differential phosphorylation of the inositol head group yields seven different phosphoinositide phosphates (PIPs) which can be mono-, bis,- or tris-phosphorylated. The roles of the cytoplasmic PIPs have been extensively studied in vesicular trafficking, ion channels, generating second messengers and, membrane and cytoskeletal dynamics. While their cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. From the nuclear PIPs, the Phosphatidylinositol 4,5-bisphosphate (PIP2) is the most abundant phosphoinositide in the cell nucleus and it participates to the nuclear architecture by regulating processes such as chromatin remodeling, DNA-damage response and gene expression. In the cell nucleus, it localizes mostly to nuclear speckles where it interacts with the splicing machinery. In nucleolus, PIP2 is involved in the RNA Polymerase I machinery to regulate rDNA transcription. Recently, we have defined a nucleoplasmic pool of PIP2 which is observed in 40 to 100nm foci. The nascent transcripts of RNA Polymerase II (RNAPII) were visualized at their periphery and RNA was shown to be...
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The role of CUP-4 protein in Wnt signalling
Žídek, Radim ; Macůrková, Marie (advisor) ; Pospíchalová, Vendula (referee)
Wnt signalling is indispensible for proper development of organisms and maintaining of adult tissue homeostasis. Its disruption often leads to disease. In nematode Caenorhabditis elegans, Wnt signalling governs vast array of developmental processes, among others also migration of the Q neuroblasts and their descendants. The sole Wnt acting in this process, EGL-20, triggers the canonical β-catenin Wnt signal transduction pathway in QL but not in QR which leads to QL remaining in the posterior while the QR migrates anteriorly. This represents a useful tool for studying Wnt signalling. Recently, mutation of gene cup-4 was found to disrupt migration of the QL neuroblast in a small proportion of the mutant population. cup-4 encodes a ligand-gated ion channel family homologue and it was shown to participate in endocytosis by coelomocytes, specialized phagocytic cells in the C. elegans body cavity. Here, I present the results of my effort to determine the place of CUP-4 action in Wnt signalling and to elucidate the mechanism of its function. I found that CUP-4 acts upstream of PRY- 1/Axin, which is involved in signal transduction in signal receiving cells, and most probably downstream of adaptin AP2, which is important for recycling of Wnt cargo receptor Wntless (Wls) in Wnt producing cell. cup-4 also...
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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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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Phosphoinositides and their effectors in regulation of the Wnt signalling pathway
Knop, Filip ; Macůrková, Marie (advisor) ; Krausová, Michaela (referee)
Phosphoinositides (PIs) make up only a small proportion of overall amount of lipids in cell membranes. However, their function mediated through protein effectors is indispensable for cell signaling, vesicular trafficking, cell movement and other important aspects of cellular life. In this bachelor thesis function of PIs is described in relation to Wnt signaling pathway. Proper execution of several steps of the Wnt signaling pathway requires the presence of PIs. Retrograde transport of Wntless (Wls) from the plasma membrane (PM) back to the Golgi apparatus (GA) in Wnt producing cells or internalization of Wnt receptors in Wnt receiving cells are only two examples. All processes are tightly regulated and malfunction of enzymes processing PIs can cause their deregulation resulting in disruption of the Wnt signaling pathway. As deregulated Wnt signaling is a known cause of serious diseases including cancer, understanding the crosstalk between PIs and Wnt signaling could help in designing novel strategies for therapeutic intervention.
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Mechanismy regulace aktivity proteinu MTM-6 na endosomech.
Horázná, Monika ; Macůrková, Marie (advisor) ; Fafílek, Bohumil (referee)
Wnt signalling belongs to conserved pathways and mediates cell fate decision, development, regeneration and adult tissue homeostasis. Disruption or misregulation of Wnt signalling pathway often leads to disease. Wnt proteins are hydrophobic glycoproteins which need a special receptor for transport from Golgi Apparatus to cell surface, which is called MIG-14 in Caenorhabditis elegans and Wntless (Wls) in mammals. In this study, I focus on understanding mechanisms that regulate MTM-6 protein activity. MTM-6, a lipid phosphatase associated with endosomal membrane, has been recently identified as a regulator of MIG-14/Wls trafficking in Caenorhabditis elegans. Silencing of mtm-6 leads to misregulation of some Wnt-directed processes, such as migration of Q neuroblasts progeny. This study reports identification of novel mtm-6 genetic interactors that have been found to influence migration of Q neuroblasts progeny through Wnt signalling. New knowledge about mtm-6 genetic interactions bring us near to understanding of Wnt signalling regulation. Keywords: Caenorhabditis elegans, MTM-6, SEL-5, Wntless, Wnt, endosomes, phosphoinositides, retromer
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The role of CUP-4 protein in Wnt signalling
Žídek, Radim ; Macůrková, Marie (advisor) ; Pospíchalová, Vendula (referee)
Wnt signalling is indispensible for proper development of organisms and maintaining of adult tissue homeostasis. Its disruption often leads to disease. In nematode Caenorhabditis elegans, Wnt signalling governs vast array of developmental processes, among others also migration of the Q neuroblasts and their descendants. The sole Wnt acting in this process, EGL-20, triggers the canonical β-catenin Wnt signal transduction pathway in QL but not in QR which leads to QL remaining in the posterior while the QR migrates anteriorly. This represents a useful tool for studying Wnt signalling. Recently, mutation of gene cup-4 was found to disrupt migration of the QL neuroblast in a small proportion of the mutant population. cup-4 encodes a ligand-gated ion channel family homologue and it was shown to participate in endocytosis by coelomocytes, specialized phagocytic cells in the C. elegans body cavity. Here, I present the results of my effort to determine the place of CUP-4 action in Wnt signalling and to elucidate the mechanism of its function. I found that CUP-4 acts upstream of PRY- 1/Axin, which is involved in signal transduction in signal receiving cells, and most probably downstream of adaptin AP2, which is important for recycling of Wnt cargo receptor Wntless (Wls) in Wnt producing cell. cup-4 also...
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