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The role of ARP2/3 complex in plant cells
Schiebertová, Petra ; Schwarzerová, Kateřina (advisor) ; Martinec, Jan (referee)
2 Abstract ARP2/3 protein complex is formed from seven proteins (ARP2, ARP3 and ARC1- ARPC5) with a relatively conserved structure. ARP2/3 complex branches and nucleates new actin filaments. This thesis focuses on the study of the role and importance of the individual subunits of the complex ARP2/3 in plants. One of the principal aims of this work is to determine whether complex ARP2/3 may at least partially maintain its role when one or more of the subunits are not available. Furthermore if the individual subunits play another, plant-specific role and if the subunits are functionally equivalent in the complex. The main way how to achieve this objective is the analysis of multiple mutants of Arabidopsis thaliana in subunits of ARP2/3 complex. After comparing several phenotypes of mutant lines it is obvious that all the subunits are functionally equivalent. A loss of ARPC5 subunit usually manifests the strongest phenotypic expression. On the contrary, loss ARPC3 and ARPC2b subunits have weak phenotypic manifestations. Because some phenotypes, such as phenotype distorted trichomes was detected only in some mutant lines, whereas the phenotype of faster roots gravitropic response or vacuolar system fragmentation that was detected in all analyzed mutants suggests, that different subunits play varying roles...
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Role of cytoskeleton in plant cell morphogenesis
Miklánková, Pavlína ; Schwarzerová, Kateřina (advisor) ; Sekereš, Juraj (referee)
The cells are able to acquire variety of shapes, in which cytoskeleton plays an important role. Cytoskeleton influences deposition of cell wall materials, regulates vesicle movement in cell, participates in exocytosis and endocytosis. Cortical microtubules affect celulose accumulation in cell wall and determine direction of cell expansion, although the exact connection between microtubules and cellulose remains unclear. Actin promotes growth and contributes to its spatial regulation in both tip ang diffuse growing cells. Actin is important for secretion in expanding cells but its exact functions in cell growth regulation are not explained yet. Analysis of mutants, spectroscopic methods, cytoskeletal drugs, fluorescence proteins and other methods are used to better understand how actin and microtubule cytoskeleton are integrated during plant cell morphogenesis. Epidermal and trichome cells of Arabidopsis thaliana are a good model of research and they are used for most studies.
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The role of ARPC2 in plant cells
Šlajcherová, Kateřina ; Schwarzerová, Kateřina (advisor) ; Klíma, Petr (referee)
ARPC2 protein localization in a plant cell Kateřina Šlajcherová 1 Abstract Actin cytoskeleton is an ubiquitous structure which plays numerous irreplacable roles. Actin nucleation is, beside formins, performed by ARP2/3 complex (actin-related protein), comprising of seven subunits (ARP2, 3, C1-C5) and activated by protein SCAR/WAVE complex. ARP2/3 complex is attached to the membrane and branches existing microfilaments, apart from nucleating them de novo. ARP2/3 mutants in most organisms show severe defects. However, plant mutants exhibit only mild phenotype, for example, Arabidopsis thaliana ARPC2 mutant (dis2-1) has deformed trichomes and leaf epidermal cells, but its viability is not impaired. The aim of the thesis is to map ARPC2 localization within the cell and broaden our understanding of ARP2/3 complex role in plant cell morphogenesis. Tobacco ARPC2 (NtArpC2) subunit was visualized in Arabidopsis plants, using the GFP fusion protein as well as imunofluorescence and anti-ARPC2 antibody. Experiments were undertaken to collocalize the subunit with actin and microtubular cytoskeleton, with mitochondrions, endosomes and other membrane organelles. The specimens were observed in confocal and TIRF microscope. The GFP-NtARPC2 protein shows as motile dots; their movement, but not their existence, is dependent...
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Characterization of the PTEN domain of selected Arabidopsis class II formins
Přerostová, Sylva ; Cvrčková, Fatima (advisor) ; Havelková, Lenka (referee)
Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...
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Functional analysis of plant Arp2/3 complex subunits
Kukla, Jakub ; Schwarzerová, Kateřina (advisor) ; Honys, David (referee)
1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...
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Non-traditional roles of formins besides actin nucleation
Metlička, Jáchym ; Cvrčková, Fatima (advisor) ; Opatrný, Zdeněk (referee)
Formin homology 2 (FH2) domaincontaining proteins (formins) have, since their discovery in 1990, been observed in all analyzed species of eukaryotic kingdoms. Our knowledge of structure and function of the defining FH2 domain has greatly increased over the last couple of years. Its function in nucleation, polymerization and processive capping of actin filaments designates formin protein family an important cytoskeletonremodelling factor. But FH2 domain is just one part of the puzzle additional optional conserved peptide structures surrounding it, as well as concrete variation of the FH2 domain itself, greatly influence the functional properties and cellular localization of the resultant formin protein. Formins have been implicated in variety of cellular processes, which often (but not always) involve the cytoskeleton e.g. Factin network management, crosstalk of Factin filaments and microtubules or plasma membrane. They also partake in processes integral to cell division, function in conserved signalling pathways and much more. This thesis explains the structure and function of FH2 and FH1 domains, outlines the main formin phylogenetic clades in multicellular eukaryotes and reviews various roles that formins fulfill or are thought to fulfill. Such goal, however, is very bold and (considering the...
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Cloning and characterisation of selected Class II formins
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Rösel, Daniel (referee)
Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...
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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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Cryptosporidium Diversity of Selected Bat Species (Chiroptera)
HOŘICKÁ, Anna
Total 262 samples of different Chiropterans originating from 27 localities in the Czech Republic were collected. Occurrence and prevalence of Cryptosporidium infection in bats were screened using staining method and molecular tools. While no Cryptosporidium oocyst was detected by microscopy, PCR analyses revealed presence of three positive specimens from three common pipistrelle (Pipistrellus pipistrellus). Zoonotic C. parvum was detected in one case and novel Cryptosporidium bat genotype III was found in other two samples.
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