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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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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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Functional characterization of selected microtubule regulatory
Vinopal, Stanislav ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) play crucial roles in intracellular organization and transport, cell polarity, motility, signalling, division and differentiation. MTs form complex arrays, which are, due to their highly dynamic nature, capable of rapid reorganization in response to cellular requirements. Dynamics, stability and spatial organization of MTs are regulated by many factors including MT regulatory proteins. In the presented study we functionally characterized three selected MT regulatory proteins: Ca2+ -sensor STIM1, MT severing protein spastin and γ-tubulin that is essential for MT nucleation. We found out that activation of bone marrow mast cells (BMMCs) leads to the formation of plasma membrane protrusions containing MTs. Formation of these MT protrusions is dependent on an influx of extracellular Ca2+ regulated by protein STIM1, located in endoplasmic reticulum. STIM1 associates with MTs and its depletion prevents formation of MT protrusions. This indicates that Ca2+ ions might be involved in MT regulation. Since STIM1 depletion also causes defects in chemotaxis, we propose that MT protrusions might be involved in sensing of external signals recognized by BMMCs. Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumor in humans. We demonstrated that MT severing...
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Microtubule-associated proteins in plants
Benáková, Martina ; Krtková, Jana (advisor) ; Vinopal, Stanislav (referee)
1. Abstract and key words MTs are one of the basic cellular protein structure. Their features and function are influenced and modified by group of other proteins, i.e. microtubule-associated proteins (MAPs). In the last decades, an extensive research on MAPs and their wide range of functions has been carried out. Therefore we are aware of the involvement of some of the MAPs in MT dynamics, other have been shown to have rather structural function. They bundle MTs with various cell structures, such as the other MTs, proteins, organelles, actin cytoskeleton or plasma membrane. Many described MAPs are homologous in the whole eukaryotic domain, for example MAP65 or EB1 (END BINDING 1) family, therefore it is interesting to follow if and how the functions of plant MAPs differ from their animal counterparts. On the other hand, there are many specific MAPs with unique functions in plants, e.g. ATK5 or SPR1 (SPIRAL 1). This Bachelor thesis is a survey on current knowledge of plant MAPs and it makes an effort to present their characteristic and functions in plant cell and organism. Key words: cytoskeleton, microtubules, microtubule-associated proteins, plant cell, growth and development
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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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Functional characterization of selected microtubule regulatory
Vinopal, Stanislav ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) play crucial roles in intracellular organization and transport, cell polarity, motility, signalling, division and differentiation. MTs form complex arrays, which are, due to their highly dynamic nature, capable of rapid reorganization in response to cellular requirements. Dynamics, stability and spatial organization of MTs are regulated by many factors including MT regulatory proteins. In the presented study we functionally characterized three selected MT regulatory proteins: Ca2+ -sensor STIM1, MT severing protein spastin and γ-tubulin that is essential for MT nucleation. We found out that activation of bone marrow mast cells (BMMCs) leads to the formation of plasma membrane protrusions containing MTs. Formation of these MT protrusions is dependent on an influx of extracellular Ca2+ regulated by protein STIM1, located in endoplasmic reticulum. STIM1 associates with MTs and its depletion prevents formation of MT protrusions. This indicates that Ca2+ ions might be involved in MT regulation. Since STIM1 depletion also causes defects in chemotaxis, we propose that MT protrusions might be involved in sensing of external signals recognized by BMMCs. Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumor in humans. We demonstrated that MT severing...
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Microtubule-associated proteins in plants
Benáková, Martina ; Krtková, Jana (advisor) ; Vinopal, Stanislav (referee)
1. Abstract and key words MTs are one of the basic cellular protein structure. Their features and function are influenced and modified by group of other proteins, i.e. microtubule-associated proteins (MAPs). In the last decades, an extensive research on MAPs and their wide range of functions has been carried out. Therefore we are aware of the involvement of some of the MAPs in MT dynamics, other have been shown to have rather structural function. They bundle MTs with various cell structures, such as the other MTs, proteins, organelles, actin cytoskeleton or plasma membrane. Many described MAPs are homologous in the whole eukaryotic domain, for example MAP65 or EB1 (END BINDING 1) family, therefore it is interesting to follow if and how the functions of plant MAPs differ from their animal counterparts. On the other hand, there are many specific MAPs with unique functions in plants, e.g. ATK5 or SPR1 (SPIRAL 1). This Bachelor thesis is a survey on current knowledge of plant MAPs and it makes an effort to present their characteristic and functions in plant cell and organism. Key words: cytoskeleton, microtubules, microtubule-associated proteins, plant cell, growth and development
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