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Kinases regulating AP2 complex phosphorylation in Caenorhabditis elegans
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Vinopal, Stanislav (referee)
Heterotetrameric adaptor protein 2 complex (AP2) is a fundamental component of clathrin-coated pits playing a part in every step of clathrin-coated vesicle generation. Although the mechanism of AP2 function has been extensively studied for no less than 20 years, the exact role of the regulatory phosphorylation on T156 of its μ2 subunit remains unclear. The main kinase responsible for the T156 phosphorylation in mammals is AAK1; however, many observations suggest that other kinases collaborate on this event. The aim of this project is to shed light on the importance of AP2 phosphorylation in Caenorhabditis elegans and elucidate the function of SEL-5/AAK1 kinase. To determine the relationship between SEL-5/AAK1, AP2 phosphorylation, and the function of endocytosis, we used a combination of phenotype analysis of C. elegans transgenic lines and in vitro assays. We showed that DPY-23, the C. elegans μ2 subunit, is phosphorylated analogously to its human orthologue. We confirmed that DPY-23 phosphorylation depends on SEL-5, yet we were not able to prove the kinase activity of SEL-5 directly. Interestingly, our results further revealed that DPY-23 phosphorylation is dispensable for the endocytosis of a model cargo, and we also showed that interaction motifs located outside the kinase domain of SEL-5 are...
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Actin and its regulation in clathrin endocytosis
Pešanová, Denisa ; Libusová, Lenka (advisor) ; Janštová, Vanda (referee)
4 Abstract Actin filaments and their dynamics play an important role in eukaryotic membrane remodelling. Actin regulatory proteins are required for actin dynamics control. During clathrin-mediated endocytosis actin and regulatory proteins interact with clathrin and adaptors proteins. Together they assist membrane invagination and scission of vesicles. Actin structures are fundamental for formation, internalization and movement of endocytic vesicles in yeast. In mammalian cells, actin is less involved especially in the early stage of endocytosis. Models reflecting different roles of actin in clathrin-mediated endocytosis in yeast and mammals are still lacking despite the latest imaging methods. The goal of this bachelor thesis is to offer a compact summary of controversial observations of actin regulation in endocytosis based on recent studies and simultaneously present selected mechanisms of interactions of the most important proteins, which influence actin dynamics in the clathrin-mediated endocytosis. Key words actin, clathrin-mediated endocytosis, Saccharomyces cerevisiae, regulatory proteins
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Mechanisms regulating the function of adaptor protein 2 complex during endocytosis
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Pleskot, Roman (referee)
Adaptor protein complex 2 (AP2) mediates the interaction of clathrin with the plasma membrane and thus enables the clathrin-coated vesicle formation. AP2 is also responsible for cargo recognition and it recognizes cargo either directly using endocytic motifs YxxΦ or [DE]xxxL[LI] in the cytosolic domains of cargo or indirectly via additional adaptor proteins from which β-arrestin and ARH are the best-known. The binding sites for endocytic motifs are located in the core of AP2 complex and, similarly to the clathrin-binding site, they are blocked by autoinhibitory mechanism in the inactive cytosolic form of AP2. Therefore, binding of endocytic motifs and clathrin must be preceded by conformational change of AP2 complex which is triggered by membrane-bound phosphatidylinositol-4,5-bisphosphates and greatly facilitated by phosphorylation at Thr156 by AAK1 kinase. AP2 is also important for later stages of endocytosis during which it recruits proteins responsible for membrane curvature, fission, and eventual disassembly of clathrin coat. Repeated association of AP2 with the plasma membrane is prevented by the protein NECAP, but the mechanism of inactivation is still poorly understood.
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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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Actin and its regulation in clathrin endocytosis
Pešanová, Denisa ; Libusová, Lenka (advisor) ; Janštová, Vanda (referee)
4 Abstract Actin filaments and their dynamics play an important role in eukaryotic membrane remodelling. Actin regulatory proteins are required for actin dynamics control. During clathrin-mediated endocytosis actin and regulatory proteins interact with clathrin and adaptors proteins. Together they assist membrane invagination and scission of vesicles. Actin structures are fundamental for formation, internalization and movement of endocytic vesicles in yeast. In mammalian cells, actin is less involved especially in the early stage of endocytosis. Models reflecting different roles of actin in clathrin-mediated endocytosis in yeast and mammals are still lacking despite the latest imaging methods. The goal of this bachelor thesis is to offer a compact summary of controversial observations of actin regulation in endocytosis based on recent studies and simultaneously present selected mechanisms of interactions of the most important proteins, which influence actin dynamics in the clathrin-mediated endocytosis. Key words actin, clathrin-mediated endocytosis, Saccharomyces cerevisiae, regulatory proteins
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